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We have previously demonstrated that itaconic acid can regulate osteoclast differentiation in vitro and in vivo, thereby affecting the progression of osteoporosis. The role of Irg1 as itaconic acid catalytic enzyme in bone homeostasis has not been clearly elucidated. Here, we detected enhanced the osteoclast differentiation in Irg1-deficiency BMMs, along with the expression of genes associated with osteoclastogenesis. Irg1 knockout promoted the expression of Nfatc1 and F-actin ring formation, with the inhibited production of itaconate. RNA-seq analysis was carried out and we proved that Grk5 expression was increased the most. Inhibition of Grk5 attenuated the effect of Irg1 in the osteoclastogenesis. However, micro-CT analysis showed no significant difference of bone trabecular structure in Irg1 knockout mice. Moreover, we observed no significant difference of osteoclasts numbers in the femur of Irg1 knockout mice in vivo. And similar bone formation was detected between the Irg1 knockout and WT mice, indicating that irg1 had slight effect on the bone homeostasis under physiological conditions. Surprising, we detected higher level of inflammatory factors in the bone tissues of Irg1 knockout mice. Above all, we for the first time demonstrated that Irg1 knockout promoted the osteoclastogenesis via regulating the Grk5 signaling. Regulation of irg1-Grk5 axis could be effective in treating human diseases under pathological situations in the future., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Periodontitis (PDS) is a chronic inflammatory disease initiated by a dysbiosis of oral pathogenic bacterial species, such as Porphyromonas gingivalis (Pg). These bacteria can penetrate the bloodstream, releasing various endo and exotoxins that fuel the infection, and stimulate toxic inflammation in different compartments, including the brain. However, the specific mechanisms by which PDS/Pg contribute to brain disorders, such as Alzheimer's disease (AD), remain unclear. This study assessed the effects of Pg's virulence factors - lipopolysaccharide (LPS-Pg) and gingipains (gps) K (Kgp) and Rgp - on SH-SY5Y cells. Our results demonstrated that LPS-Pg activated signaling through the Toll-like receptor (TLR)-2/4 induced a significant downregulation of G protein-coupled receptor kinase 5 (GRK5). Additionally, LPS-Pg stimulation resulted in a robust increase in Tau phosphorylation (pTau) and p53 levels, while causing a marked reduction in Bcl2 and increased cell death compared to unstimulated cells (Ns). LPS-Pg also elevated inducible nitric oxide synthase (iNOS) expression, leading to oxidative damage. In cells overexpressing GRK5 via Adenovirus, LPS-Pg failed to increase iNOS and pTau levels compared to GFP control cells. High GRK5 levels also prevented the nuclear accumulation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB). Furthermore, the overexpression of a GRK5 mutant form lacking the nuclear localization signal (ΔNLS) nearly abolished LPS-Pg induced p53 and iNOS upregulation. Finally, we tested whether Kgp and Rgp mediated similar effects and our data showed that both gps caused a marked downregulation of GRK5 leading to increased p53 and pTau levels. In conclusion, this study provides further insight into the toxic effects elicited by Pg in cells and suggests that preventing GRK5 deficiency may be a valid strategy to mitigate Pg-induced toxic effects (i.e. cell death, oxidative damage, and Tau hyperphosphorylation) in SH-SY5Y cells, which are typical molecular hallmarks of neurodegenerative disorders., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Alzheimer's disease (AD) poses an immense challenge in healthcare, lacking effective therapies. This study investigates the potential of anthranilamide derivative (AAD23), a selective M2 receptor antagonist, in proactively preventing cognitive impairments and cholinergic neuronal degeneration in G protein-coupled receptor kinase-5-deficient Swedish APP (GAP) mice. GAP mice manifest cognitive deficits by 7 months and develop senile plaques by 9 months. A 6-month AAD23 treatment was initiated at 5 months and stopped at 11 months before behavioral assessments without the treatment. AAD23-treated mice exhibited preserved cognitive abilities and improved cholinergic axonal health in the nucleus basalis of Meynert akin to wildtype mice. Conversely, vehicle-treated GAP mice displayed memory deficits and pronounced cholinergic axonal swellings in the nucleus basalis of Meynert. Notably, AAD23 treatment did not alter senile plaques and microgliosis. These findings highlight AAD23's efficacy in forestalling AD-related cognitive decline in G protein-coupled receptor kinase-5-deficient subjects, attributing its success to restoring cholinergic neuronal integrity and resilience, enhancing resistance against diverse degenerative insults., Competing Interests: Conflicts of interest The authors declare they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

G protein-coupled receptors (GPCRs) are key modulators of cell signaling. Multiple GPCRs are present in the heart where they regulate cardiac homeostasis including processes such as myocyte contraction, heart rate and coronary blood flow. GPCRs are pharmacological targets for several cardiovascular disorders including heart failure (HF) such as beta-adrenergic receptor (βAR) blockers and angiotensin II receptor (AT1R) antagonists. The activity of GPCRs are finely regulated by GPCR kinases (GRKs), which phosphorylate agonist-occupied receptors and start the process of desensitization. Among the seven members of the GRK family, GRK2 and GRK5 are predominantly expressed in the heart, where they exhibit both canonical and non-canonical functions. Both kinases are known to be increased in cardiac pathologies and contribute to pathogenesis through their roles in different cellular compartments. Lowering or inhibiting their actions mediate cardioprotective effects against pathological cardiac growth and failing heart. Therefore, given their importance in cardiac dysfunction, these kinases are drawing attention as promising targets for the treatment of HF, which needs improved therapies. Over the past three decades, broad knowledge on GRK inhibition in HF has been gained by studies using genetically engineered animal models or through gene therapy with peptide inhibitors or using small molecule inhibitors. In this mini review, we summarize the work focusing on GRK2 and GRK5 but also discuss a couple of the non-abundant cardiac subtypes and their multi-functional roles in the normal and diseased heart and the potential and therapeutic targets., (© 2023 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have influenced the work reported in this paper., (Copyright © 2022. Published by Elsevier Masson SAS.)

Triple-negative breast cancer (TNBC) is a malignant tumor that threatens women's health. Exploring novel development-associated biomarkers could help improve the survival rate of TNBC. This study evaluated the significance and mechanism of LINC01315 in TNBC progression aiming to identify a potential biomarker. There were 103 TNBC patients that provided clinical tissues in this study. The expression of LINC01315 was assessed by PCR and its association with clinical data was evaluated by statistical analyses. The in vitro cell experiments were conducted to estimate the biological effect of LINC01315 and its molecular mechanism. A significant upregulation of LINC01315 was observed in TNBC, which was associated with disease development and severity of patients. The upregulation of LINC01315 could be a symptom of the poor prognosis of patients. The knockdown of LINC01315 suppressed the main cellular processes of TNBC progression. Additionally, miR-876-5p was demonstrated to be a target of LINC01315 and regulate the expression of GRK5, through which LINC01315 modulated the progression of TNBC. Upregulated LINC01315 in TNBC indicated the malignant development and poor survival rate of patients. Inhibition of LINC01315 might be a potential therapeutic strategy of TNBC.

Signaling-biased ligands acting on G-protein-coupled receptors (GPCRs) differentially activate heterotrimeric G proteins and β-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 β-arrestin conformation and function. Using the angiotensin II (Ang II) type-1 receptor (AT1R), we show that β-arrestin recruitment depends on both GRK2/3 and GRK5/6 upon binding of Ang II, but solely on GRK5/6 upon binding of the β-arrestin-biased ligand TRV027. With pharmacological inhibition or genetic loss of Gq, GRK-subtype selectivity and β-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 β-arrestin functions., (© 2022. The Author(s).)

Aims: Myocardial infarction (MI) is the most common cause of heart failure (HF) worldwide. G protein-coupled receptor kinase 5 (GRK5) is upregulated in failing human myocardium and promotes maladaptive cardiac hypertrophy in animal models. However, the role of GRK5 in ischemic heart disease is still unknown. In this study, we evaluated whether myocardial GRK5 plays a critical role post-MI in mice and included the examination of specific cardiac immune and inflammatory responses., Methods and Results: Cardiomyocyte-specific GRK5 overexpressing transgenic mice (TgGRK5) and non-transgenic littermate control (NLC) mice as well as cardiomyocyte-specific GRK5 knockout mice (GRK5cKO) and wild type (WT) were subjected to MI and, functional as well as structural changes together with outcomes were studied. TgGRK5 post-MI mice showed decreased cardiac function, augmented left ventricular dimension and decreased survival rate compared to NLC post-MI mice. Cardiac hypertrophy and fibrosis as well as fetal gene expression were increased post-MI in TgGRK5 compared to NLC mice. In TgGRK5 mice, GRK5 elevation produced immuno-regulators that contributed to the elevated and long-lasting leukocyte recruitment into the injured heart and ultimately to chronic cardiac inflammation. We found an increased presence of pro-inflammatory neutrophils and macrophages as well as neutrophils, macrophages and T-lymphocytes at 4-days and 8-weeks respectively post-MI in TgGRK5 hearts. Conversely, GRK5cKO mice were protected from ischemic injury and showed reduced early immune cell recruitment (predominantly monocytes) to the heart, improved contractility and reduced mortality compared to WT post-MI mice. Interestingly, cardiomyocyte-specific GRK2 transgenic mice did not share the same phenotype of TgGRK5 mice and did not have increased cardiac leukocyte migration and cytokine or chemokine production post-MI., Conclusions: Our study shows that myocyte GRK5 has a crucial and GRK-selective role on the regulation of leucocyte infiltration into the heart, cardiac function and survival in a murine model of post-ischemic HF, supporting GRK5 inhibition as a therapeutic target for HF., (© The Author(s) 2021. Published by Oxford University Press on behalf of the European Society of Cardiology.)

G protein coupled receptor kinase 5 (GRK5) is localized within the nucleus and moderates functions such as DNA transcription, in addition to its localization at the plasma membrane. In this report, we show that GRK5 modifies the nucleolar stress response activated by the DNA polymerase inhibitor, actinomycin D (ActD). We show an increased sensitivity to the apoptotic effects of ActD on cervical HeLa cells and the breast cancer cell line MDA MB 231 with reduced protein expression of GRK5. We also tested two types of breast cancer cells (MDA MB 231 and MCF7 cells) and found that the rate of response to ActD varied between them because they have innate differences in the protein expression of GRK5. We also found that GRK5 phosphorylates nucleophosmin (NPM1) at T199 before and during the early stages of ActD treatment. Phosphorylation at T199 increases the ability of NPM1 to interact with p14 ARF in vitro, which may affect the protein expression levels of p14 ARF . We found that the expression levels of p14 ARF were lower in the cells transfected with the control shRNA, but higher in cells transfected with GRK5 shRNA. Collectively, this suggests that GRK5 modifies the nucleolar stress response associated with ActD.

Due to extremely poor systemic bioavailability, the mechanism by which curcumin increases energy expenditure remains unelucidated. Accumulating evidence suggests a strong association between the gut microbiota (GM) and energy metabolism. We investigated whether the GM mediates the effects of curcumin on improving energy homeostasis. High-fat diet (HFD)-fed wild type, uncoupling protein 1 (Ucp1) knockout and G protein-coupled membrane receptor 5 (TGR5) knockout mice were treated with curcumin (100 mg kg -1 d -1 , p.o.). Curcumin-treated HFD-fed mice displayed decreased body weight gain and augmented cold tolerance due to enhanced adaptive thermogenesis as compared with that in control mice. The anti-obesity effects of curcumin were abolished by Ucp1 knockout. 16S ribosomal DNA sequencing analysis revealed that curcumin restructured the GM in HFD-fed mice. Fecal microbiota transplantation (FMT) and endogenous GM depletion indicated that the GM mediated the enhanced effect of curcumin on Ucp1-dependent thermogenesis. Curcumin altered bile acid (BA) metabolism with increased fractions of circulating deoxycholic acid (DCA) and lithocholic acid (LCA), which are the two most potent ligands for TGR5. Consistently, the enhanced effect of curcumin on Ucp1-dependent thermogenesis was eliminated by TGR5 knockout. Curcumin requires the GM and TGR5 to activate the cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) signaling pathway in thermogenic adipose tissue. Here, we demonstrated that the GM mediates the effects of curcumin on enhancing Ucp1-dependent thermogenesis and ameliorating HFD-induced obesity by influencing BA metabolism. We disclosed the potential of nutritional and pharmacologic manipulations of the GM to enhance Ucp1-dependent thermogenesis in the prevention and treatment of obesity.

We have previously reported that the repeated exposure to cannabinoids upregulates and enhances the activity of serotonin 2A (5-HT 2A ) and dopamine 2 (D 2 ) receptors and facilitates the formation of D 2 -5-HT 2A receptor heterodimers in the rat prefrontal cortex and two neuronal cell lines. Because the repeated exposure to cannabinoids has been associated with adverse neuropsychiatric disorders, this study investigated the mechanisms that underly the cannabinoid-mediated regulation of D 2 receptor expression in a neuronal cell model, CLU213 cells. We initially tested the effects of repeated exposure (72 h) to a non-selective cannabinoid agonist (1 nM CP55940), a selective CB 1 receptor agonist (15 nM ACEA), or a selective CB 2 receptor drug (1 nM GP1a) on the expression of postsynaptic D 2 (D 2 L) receptors in CLU213 cells. Repeated CP55940, GP1a, or ACEA treatments significantly increased D 2 L receptor protein levels (99 % ± 7%, 30 % ± 7%, and 39 % ± 5% increases compared with control levels, respectively). Repeated exposure to both GP1a and ACEA increased D 2 L receptor protein levels by 73 % ± 8%. Interestingly, CP55940 and GP1a, but not ACEA, upregulated D 2 mRNA. Using cells that were stably transfected with short-hairpin RNA (shRNA) lentiviral particles targeting CB 2 receptors, G protein-coupled receptor kinase 5 (GRK5), and β-arrestin 2, we found that CB 2 receptors regulated D 2 expression through a mechanism that is dependent on GRK5, β-arrestin 2, and extracellular signal-related kinase (ERK)1/2. We also found that repeated exposure to either ACEA or GP1a selectively stimulated the protein and mRNA expression of GRK proteins. ACEA significantly upregulated GRK2 proteins, whereas GP1a upregulated GRK5 protein expression. Our results identified mechanisms associated with the upregulation of D 2 receptors in neuronal cells after the repeated exposure to cannabinoids. These data can shed light on the mechanisms that can be targeted to prevent potential adverse effects, while simultaneously determining the therapeutic benefits of cannabinoids., (Copyright © 2021 Elsevier B.V. All rights reserved.)

G protein-coupled receptor kinases (GRKs), in addition to their role in modulating signal transduction mechanisms associated with activated G protein-coupled receptors (GPCRs), can also interact with many non-GPCR proteins to mediate cellular responses to chemotherapeutics. The rationale for this study is based on the presumption that GRK2 modulates the responses of cancer cells to the chemotherapeutic cisplatin. In this report, we show that GRK2 modulates the responses of cancer cells to cisplatin. Cervical cancer HeLa cells stably transfected with GRK2 shRNA, to decrease GRK2 protein expression, show increased sensitivity to cisplatin. Of interest, these cells also show increased accumulation of NADPH, associating with decreased NADP buildup, at low concentrations of cisplatin tested. These changes in NADPH and NADP levels are also observed in the breast cancer MDA MB 231 cells, which has lower endogenous GRK2 protein expression levels, but not BT549, a breast cancer cell line with higher GRK2 protein expression. This effect of NADPH accumulation may be associated with a decrease in NADPH oxidase 4 (NOX4) protein expression, which is found to correlate with GRK2 protein expression in cancer cells-a relationship which mimics that observed in cardiomyocytes. Furthermore, like in cardiomyocytes, GRK2 and NOX4 interact to form complexes in cancer cells. Collectively, these results suggest that GRK2 interacts with NOX4 to modify cisplatin sensitivity in cancer cells and may also factor into the success of cisplatin-based regimens.

G protein-coupled receptors (GPCRs) are the largest family of cell-surface receptors and they are responsible for the transduction of extracellular signals, regulating almost all aspects of mammalian physiology. These receptors are specifically regulated by a family of serine/threonine kinases, called GPCR kinases (GRKs). Given the biological role of GPCRs, it is not surprising that GRKs are also involved in several pathophysiological processes. Particular importance is emerging for GRK5, which is a multifunctional protein, expressed in different cell types, and it has been found located in single or multiple subcellular compartments. For instance, when anchored to the plasma membrane, GRK5 exerts its canonical function, regulating GPCRs. However, under certain conditions (e.g., pro-hypertrophic stimuli), GRK5 translocates to the nucleus of cells where it can interact with non-GPCR-related proteins as well as DNA itself to promote "non-canonical" signaling, including gene transcription. Importantly, due to these actions, several studies have demonstrated that GRK5 has a pivotal role in the pathogenesis of chronic-degenerative disorders. This is true in the cardiac cells, tumor cells, and neurons. For this reason, in this review article, we will inform the readers of the most recent evidence that supports the importance of targeting GRK5 to prevent the development or progression of cancer, cardiovascular, and neurological diseases.

Pathological remodeling of the heart is a hallmark of chronic heart failure (HF) and these structural changes further perpetuate the disease. Cardiac fibroblasts are the critical cell type that is responsible for maintaining the structural integrity of the heart. Stress conditions, such as a myocardial infarction (MI), can activate quiescent fibroblasts into synthetic and contractile myofibroblasts. G protein-coupled receptor kinase 5 (GRK5) is an important mediator of cardiovascular homeostasis through dampening of GPCR signaling, and is expressed in the heart and up-regulated in human HF. Of note, GRK5 has been demonstrated to translocate to the nucleus in cardiomyocytes in a calcium-calmodulin (Ca 2+ -CAM)-dependent manner, promoting hypertrophic gene transcription through activation of nuclear factor of activated T cells (NFAT). Interestingly, NFAT is also involved in fibroblast activation. GRK5 is highly expressed and active in cardiac fibroblasts; however, its pathophysiological role in these crucial cardiac cells is unknown. We demonstrate using adult cardiac fibroblasts that genetic deletion of GRK5 inhibits angiotensin II (AngII)-mediated fibroblast activation. Fibroblast-specific deletion of GRK5 in mice led to decreased fibrosis and cardiac hypertrophy after chronic AngII infusion or after ischemic injury compared to nontransgenic littermate controls (NLCs). Mechanistically, we show that nuclear translocation of GRK5 is involved in fibroblast activation. These data demonstrate that GRK5 is a regulator of fibroblast activation in vitro and cardiac fibrosis in vivo. This adds to previously published data which demonstrate the potential beneficial effects of GRK5 inhibition in the context of cardiac disease., Competing Interests: The authors declare no competing interest., (Copyright © 2021 the Author(s). Published by PNAS.)

The phosphorylation of G protein-coupled receptors (GPCRs) by GPCR kinases (GRKs) facilitates arrestin binding and receptor desensitization. Although this process can be regulated by Ca 2+ -binding proteins such as calmodulin (CaM) and recoverin, the molecular mechanisms are poorly understood. Here, we report structural, computational, and biochemical analysis of a CaM complex with GRK5, revealing how CaM shapes GRK5 response to calcium. The CaM N and C domains bind independently to two helical regions at the GRK5 N and C termini to inhibit GPCR phosphorylation, though only the C domain interaction disrupts GRK5 membrane association, thereby facilitating cytoplasmic translocation. The CaM N domain strongly activates GRK5 via ordering of the amphipathic αN-helix of GRK5 and allosteric disruption of kinase-RH domain interaction for phosphorylation of cytoplasmic GRK5 substrates. These results provide a framework for understanding how two functional effects, GRK5 activation and localization, can cooperate under control of CaM for selective substrate targeting by GRK5., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

β-adrenergic receptors (β-ARs) play a major role in the physiological regulation of cardiac function through signaling routes tightly controlled by G protein-coupled receptor kinases (GRKs). Although the acute stimulation of β-ARs and the subsequent production of cyclic AMP (cAMP) have beneficial effects on cardiac function, chronic stimulation of β-ARs as observed under sympathetic overdrive promotes the development of pathological cardiac remodeling and heart failure (HF), a leading cause of mortality worldwide. This is accompanied by an alteration in cAMP compartmentalization and the activation of the exchange protein directly activated by cAMP 1 (Epac1) signaling. Among downstream signals of β-ARs, compelling evidence indicates that GRK2, GRK5, and Epac1 represent attractive therapeutic targets for cardiac disease. Here, we summarize the pathophysiological roles of GRK2, GRK5, and Epac1 in the heart. We focus on their signalosome and describe how under pathological settings, these proteins can cross-talk and are part of scaffolded nodal signaling systems that contribute to a decreased cardiac function and HF development.

Rationale: Cardiotoxic β 1 adrenergic receptor (β 1 AR)-CaMKII (calmodulin-dependent kinase II) signaling is a major and critical feature associated with development of heart failure. SAP97 (synapse-associated protein 97) is a multifunctional scaffold protein that binds directly to the C-terminus of β 1 AR and organizes a receptor signalosome., Objective: We aim to elucidate the dynamics of β 1 AR-SAP97 signalosome and its potential role in chronic cardiotoxic β 1 AR-CaMKII signaling that contributes to development of heart failure., Methods and Results: The integrity of cardiac β 1 AR-SAP97 complex was examined in heart failure. Cardiac-specific deletion of SAP97 was developed to examine β 1 AR signaling in aging mice, after chronic adrenergic stimulation, and in pressure overload hypertrophic heart failure. We show that the β 1 AR-SAP97 signaling complex is reduced in heart failure. Cardiac-specific deletion of SAP97 yields an aging-dependent cardiomyopathy and exacerbates cardiac dysfunction induced by chronic adrenergic stimulation and pressure overload, which are associated with elevated CaMKII activity. Loss of SAP97 promotes PKA (protein kinase A)-dependent association of β 1 AR with arrestin2 and CaMKII and turns on an Epac (exchange protein directly activated by cAMP)-dependent activation of CaMKII, which drives detrimental functional and structural remodeling in myocardium. Moreover, we have identified that GRK5 (G-protein receptor kinase-5) is necessary to promote agonist-induced dissociation of SAP97 from β 1 AR. Cardiac deletion of GRK5 prevents adrenergic-induced dissociation of β 1 AR-SAP97 complex and increases in CaMKII activity in hearts., Conclusions: These data reveal a critical role of SAP97 in maintaining the integrity of cardiac β 1 AR signaling and a detrimental cardiac GRK5-CaMKII axis that can be potentially targeted in heart failure therapy. Graphical Abstract: A graphical abstract is available for this article.

Tachykinin signaling system is present in both vertebrates and invertebrates, and functions as neuromodulator responsible for the regulation of various physiological processes. In human, the internalization of G protein-coupled receptors has been extensively characterized; however, the insect GPCR internalization has been rarely investigated. Here, we constructed two expression vectors of Bombyx tachykinin-related peptide receptor (BmTKRPR) fused with Enhanced Green Fluorescent Protein (EGFP) at the C-terminal end for direct visualization of receptor expression, localization, and trafficking in cultured mammalian HEK293 and insect Sf21 cells. Our results demonstrated that agonist-activated BmTKRPR underwent rapid internalization in a dose-and time-dependent manner via a clathrin-dependent pathway in both HEK293 and Sf21 cells. Further investigation via RNAi or specific inhibitors, or co-immunoprecipitation demonstrated that agonist-induced BmTKRPR internalization was mediated by PKC, GRK5 and β-arrestin2/BmKurtz. In addition, we also observed that most of the internalized BmTKRP receptors were recycled to the cell surface via early endosomes upon peptide ligand removal. Our study provides the first in-depth information on mechanisms underlying insect TKRP receptor internalization and perhaps aids in the interpretation of the signaling in the regulation of physiological processes., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Aldosterone (Aldo), when overproduced, is a cardiotoxic hormone underlying heart failure and hypertension. Aldo exerts damaging effects via the mineralocorticoid receptor (MR) but also activates the antiapoptotic G protein-coupled estrogen receptor (GPER) in the heart. G protein-coupled receptor (GPCR)-kinase (GRK)-2 and -5 are the most abundant cardiac GRKs and phosphorylate GPCRs as well as non-GPCR substrates. Herein, we investigated whether they phosphorylate and regulate cardiac MR and GPER. To this end, we used the cardiomyocyte cell line H9c2 and adult rat ventricular myocytes (ARVMs), in which we manipulated GRK5 protein levels via clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 and GRK2 activity via pharmacological inhibition. We report that GRK5 phosphorylates and inhibits the cardiac MR whereas GRK2 phosphorylates and desensitizes GPER. In H9c2 cardiomyocytes, GRK5 interacts with and phosphorylates the MR upon β 2 -adrenergic receptor (AR) activation. In contrast, GRK2 opposes agonist-activated GPER signaling. Importantly, GRK5-dependent MR phosphorylation of the MR inhibits transcriptional activity, since aldosterone-induced gene transcription is markedly suppressed in GRK5-overexpressing cardiomyocytes. Conversely, GRK5 gene deletion augments cardiac MR transcriptional activity. β 2 AR-stimulated GRK5 phosphorylates and inhibits the MR also in ARVMs. Additionally, GRK5 is necessary for the protective effects of the MR antagonist drug eplerenone against Aldo-induced apoptosis and oxidative stress in ARVMs. In conclusion, GRK5 blocks the cardiotoxic MR-dependent effects of Aldo in the heart, whereas GRK2 may hinder beneficial effects of Aldo through GPER. Thus, cardiac GRK5 stimulation (e.g., via β 2 AR activation) might be of therapeutic value for heart disease treatment via boosting the efficacy of MR antagonists against Aldo-mediated cardiac injury.

G protein-coupled receptors (GPCRs) are the largest family of cell-surface receptors in humans and regulate numerous physiological processes through the activation of heterotrimeric G proteins. GPCR kinases (GRKs) selectively phosphorylate active GPCRs, which promotes arrestin binding, receptor internalization, and initiation of alternative signaling pathways. GRK5 is a representative member of one of three GRK subfamilies that does not need post-translational lipidation or other binding partners to exhibit full activity against GPCRs, rendering it a useful tool for biophysical studies directed at characterizing GRK function. However, recombinant expression of GRK5 has thus far been limited to insect and mammalian systems. Here, we describe the expression of functional GRK5 in E. coli and its purification and biochemical characterization. Bacterially expressed GRK5 is hyperphosphorylated, primarily in regions known to be flexible from prior crystal structures, which slightly decreases its catalytic activity toward receptor substrates. Mutation of a single phosphorylation site, Thr10, restores kinetic parameters to those of GRK5 purified from insect cells. Consequently, bacterial expression will allow for production of GRK5 at a reduced cost and faster pace and would facilitate production of isotopically labeled kinase for NMR studies or for the incorporation of unnatural amino acids., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Objective: NF-κB-dependent signaling is an important modulator in osteoarthritis (OA), and G protein-coupled receptor kinase 5 (GRK5) regulates the NF-κB pathway. This study was undertaken to investigate the functional involvement of GRK5 in OA pathogenesis., Methods: GRK5 expression in normal and OA human knee joints was analyzed immunohistochemically. Gain- or loss-of-function experiments were performed using human and mouse chondrocytes. OA was induced in GRK5-knockout mice by destabilization of the medial meniscus, and histologic examination was performed. OA was also induced in wild-type mice, which were then treated with an intraarticular injection of amlexanox, a selective GRK5 inhibitor, every 5 days for 8 weeks., Results: GRK5 protein expression was increased in human OA cartilage. In vitro, expression levels of OA-related factors and NF-κB transcriptional activation were down-regulated by suppression of the GRK5 gene in human OA chondrocytes (3.49-fold decrease in IL6 [P < 0.01], 2.43-fold decrease in MMP13 [P < 0.01], and 2.66-fold decrease in ADAMTS4 [P < 0.01]). Conversely, GRK5 overexpression significantly increased the expression of OA-related catabolic mediators and NF-κB transcriptional activation. On Western blot analysis, GRK5 deletion reduced IκBα phosphorylation (up to 4.4-fold decrease [P < 0.05]) and decreased p65 nuclear translocation (up to 6.4-fold decrease [P < 0.01]) in mouse chondrocytes. In vivo, both GRK5 deletion and intraarticular amlexanox protected mouse cartilage against OA., Conclusion: Our results suggest that GRK5 regulates cartilage degradation through a catabolic response mediated by NF-κB signaling, and is a potential target for OA treatment. Furthermore, amlexanox may be a major compound in relevant drugs., (© 2019, American College of Rheumatology.)

The Raf kinase inhibitor protein (RKIP) activates β-adrenoceptors (β-AR) and thereby induces a well-tolerated cardiac contractility and prevents heart failure in mice. Different to RKIP-mediated β-AR activation, chronic activation of β-AR by catecholamines was shown to be detrimental for the heart. RKIP is an endogenous inhibitor of G protein coupled receptor kinase 2 (GRK2); it binds GRK2 and thereby inhibits GRK2 mediated β-AR phosphorylation and desensitization. Here, we evaluate RKIP-mediated effects on β-AR to explore new strategies for β-AR modulation. Co-immunoprecipitation assays and pull-down assays revealed subtype specificity of RKIP for the cardiac GRK isoforms GRK2 and GRK3 - not GRK5 - as well as several RKIP binding sites within their N-termini (GRK2 1-185 and GRK3 1-185 ). Overexpression of these N-termini prevented β 2 -AR phosphorylation and internalization, subsequently increased receptor signaling in HEK293 cells and cardiomyocyte contractility. Co-immunoprecipitation assays of β 2 -AR with these N-terminal GRK fragments revealed a direct interaction suggesting a steric interference of the fragments with the functional GRK-receptor interaction. Altogether, N-termini of GRK2 and GRK3 efficiently simulate RKIP effects on β-AR signaling in HEK293 cells and in cardiomyocytes by their binding to β 2 -AR and, thus, provide important insights for the development of new strategies to modulate β 2 -AR signaling., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

G protein-coupled receptor kinases (GRKs) phosphorylate the activated forms of G protein-coupled receptors (GPCRs), leading to receptor desensitization and internalization. In addition, GRKs can modify the activity of many non-GPCR-signaling pathways as well, controlling other cellular functions beyond that directly associated with a GPCR. In this report, we show that cervical cancer HeLa cells and breast cancer MDA MB 231 cells with reduced GRK5 expression display increased sensitivity to the apoptotic effects of paclitaxel (Taxol). This effect in cancer cells with low GRK5 levels could be because of blunted histone deacetylase 6 (HDAC6) activity that leads to an increase in α-tubulin acetylation levels, which augments paclitaxel sensitivity. We demonstrate that GRK5 and HDAC6 form a signaling complex in cells and in vitro. GRK5 phosphorylates HDAC6 at Ser-21 to promote its deacetylase activity. Therefore, the GRK5-HDAC6 interaction may contribute to paclitaxel resistance in cancer cells.

G protein-coupled receptors (GPCRs) are critical cellular sensors that mediate numerous physiological processes. In the heart, multiple GPCRs are expressed on various cell types, where they coordinate to regulate cardiac function by modulating critical processes such as contractility and blood flow. Under pathological settings, these receptors undergo aberrant changes in expression levels, localization and capacity to couple to downstream signalling pathways. Conventional therapies for heart failure work by targeting GPCRs, such as β-adrenergic receptor and angiotensin II receptor antagonists. Although these treatments have improved patient survival, heart failure remains one of the leading causes of mortality worldwide. GPCR kinases (GRKs) are responsible for GPCR phosphorylation and, therefore, desensitization and downregulation of GPCRs. In this Review, we discuss the GPCR signalling pathways and the GRKs involved in the pathophysiology of heart disease. Given that increased expression and activity of GRK2 and GRK5 contribute to the loss of contractile reserve in the stressed and failing heart, inhibition of overactive GRKs has been proposed as a novel therapeutic approach to treat heart failure.

Aims: Recent studies reported that cAMP-binding protein Epac1-deficient mice were protected against various forms of cardiac stress, suggesting that pharmacological inhibition of Epac1 could be beneficial for the treatment of cardiac diseases. To test this assumption, we characterized an Epac1-selective inhibitory compound and investigated its potential cardioprotective properties., Methods and Results: We used the Epac1-BRET (bioluminescence resonance energy transfer) for searching for non-cyclic nucleotide Epac1 modulators. A thieno[2,3-b]pyridine derivative, designated as AM-001 was identified as a non-competitive inhibitor of Epac1. AM-001 has no antagonist effect on Epac2 or protein kinase A activity. This small molecule prevents the activation of the Epac1 downstream effector Rap1 in cultured cells, in response to the Epac1 preferential agonist, 8-CPT-AM. In addition, we found that AM-001 inhibited Epac1-dependent deleterious effects such as cardiomyocyte hypertrophy and death. Importantly, AM-001-mediated inhibition of Epac1 reduces infarct size after mouse myocardial ischaemia/reperfusion injury. Finally, AM-001 attenuates cardiac hypertrophy, inflammation and fibrosis, and improves cardiac function during chronic β-adrenergic receptor activation with isoprenaline (ISO) in mice. At the molecular level, ISO increased Epac1-G protein-coupled receptor kinase 5 (GRK5) interaction and induced GRK5 nuclear import and histone deacetylase type 5 (HDAC5) nuclear export to promote the activity of the prohypertrophic transcription factor, myocyte enhancer factor 2 (MEF2). Inversely, AM-001 prevented the non-canonical action of GRK5 on HDAC5 cytoplasmic shuttle to down-regulate MEF2 transcriptional activity., Conclusion: Our study represents a 'proof-of-concept' for the therapeutic effectiveness of inhibiting Epac1 activity in cardiac disease using small-molecule pharmacotherapy., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2019. For permissions, please email: journals.permissions@oup.com.)

G protein-coupled receptor (GPCR) kinases (GRKs) are responsible for initiating desensitization of activated GPCRs. GRK5 is potently inhibited by the calcium-sensing protein calmodulin (CaM), which leads to nuclear translocation of GRK5 and promotion of cardiac hypertrophy. Herein, we report the architecture of the Ca 2+ ·CaM-GRK5 complex determined by small-angle X-ray scattering and negative-stain electron microscopy. Ca 2+ ·CaM binds primarily to the small lobe of the kinase domain of GRK5 near elements critical for receptor interaction and membrane association, thereby inhibiting receptor phosphorylation while activating the kinase for phosphorylation of soluble substrates. To define the role of each lobe of Ca 2+ ·CaM, we utilized the natural product malbrancheamide as a chemical probe to show that the C-terminal lobe of Ca 2+ ·CaM regulates membrane binding while the N-terminal lobe regulates receptor phosphorylation and kinase domain activation. In cells, malbrancheamide attenuated GRK5 nuclear translocation and effectively blocked the hypertrophic response, demonstrating the utility of this natural product and its derivatives in probing Ca 2+ ·CaM-dependent hypertrophy., Competing Interests: The authors declare no conflict of interest.

G protein-coupled receptor kinase 5 (GRK5) is a serine/threonine kinase whose dysfunction results in cognitive impairment and Alzheimer-like pathology, including tau hyperphosphorylation. However, the mechanisms whereby GRK5 influences tau phosphorylation remain incompletely understood. In the current study, we showed that GRK5 influenced the phosphorylation of tau via glycogen synthase kinase 3β (GSK3β). The activity of both tau and GSK3β in the hippocampus was increased in aged GRK5-knockout mice, which is consistent with what occurs in APP/PS1 transgenic mice. Furthermore, GRK5 regulated the activity of GSK3β and phosphorylated tau in vitro. Regardless of changes of GRK5 protein levels, tau hyperphosphorylation remained reduced after GSK3β activity was inhibited, suggesting that GRK5 may specifically influence tau hyperphosphorylation by modulating GSK3β activity. Taken together, our findings suggest that GRK5 deficiency contributes to the pathogenesis of Alzheimer's disease by influencing the hyperphosphorylation of tau through the activation of GSK3β., (© 2018 Wiley Periodicals, Inc.)

We synthesized a previously identified β-tubulin-derived G protein-coupled receptor kinase 2 (GKR2) peptide (GR-11-1; DEMEFTEAESNMN) and its amino-terminal extension (GR-11-1-N; GEGMDEMEFTEAESNMN) and carboxyl-terminal extension (GR-11-1-C; DEMEFTEAESNMNDLVSEYQ) peptides with the aim of finding a high-affinity peptide substrate for GRK2. GR-11-1-C showed high affinity for GRK2, but very low affinity for GKR5. Its specificity and sensitivity for GKR2 were greater than those of GR-11-1 and GR-11-1-N. These findings should be useful in designing tools for probing GKR2-mediated intracellular signaling pathways, as well as GRK2-specific drugs.

Introduction: One in every four deaths in the United States is attributed to cardiovascular disease, hence the development and employment of novel and effective therapeutics are necessary to improve the quality of life and survival of affected patient. Pathological hypertrophy is a maladaptive response by the heart to relieve wall stress that could result from cardiovascular disease. Maladaptive hypertrophy can lead to further disease progression and complications such as heart failure; hence, efforts to target hypertrophy to prevent and treat further morbidity and mortality are necessary. Areas covered: This review summarizes the compelling literature that describes the mechanistic role of GRK2 and GRK5 in maladaptive cardiac hypertrophy; it examines the approaches to inhibit these kinases in hypertrophic animal models and furthermore, it assesses the potential of GRK2 and GRK5 as therapeutic targets for hypertrophy. Expert opinion: GRK2 and GRK5 are novel therapeutic targets for pathological hypertrophy and may have added benefits of ameliorating morbidity and mortality. Despite the lesser researched role of GRK2 in cardiac hypertrophy, it may be the advantageous strategy for treating cardiac hypertrophy because of its role in other maladaptive pathways. Anti-GRK2 therapy optimization and the discovery and development of specific GRK2 and GRK5 small-molecule inhibitors is necessary for the eventual application of successful, effective therapeutics.

PH domain leucine-rich repeat protein phosphatase (PHLPP) is a serine/threonine phosphatase that has been shown to regulate cell growth and survival through dephosphorylation of several members of the AGC family of kinases. G-protein-coupled receptor kinase 5 (GRK5) is an AGC kinase that regulates phenylephrine (PE)-induced cardiac hypertrophy through its noncanonical function of directly targeting proteins to the nucleus to regulate transcription. Here we investigated the possibility that the PHLPP2 isoform can regulate GRK5-induced cardiomyocyte hypertrophy in neonatal rat ventricular myocytes (NRVMs). We show that removal of PHLPP2 by siRNA induces hypertrophic growth of NRVMs as measured by cell size changes at baseline, potentiated PE-induced cell size changes, and re-expression of fetal genes atrial natriuretic factor and brain natriuretic peptide. Endogenous GRK5 and PHLPP2 were found to interact in NRVMs, and PE-induced nuclear accumulation of GRK5 was enhanced upon down-regulation of PHLPP2. Conversely, overexpression of PHLPP2 blocked PE-induced hypertrophic growth, re-expression of fetal genes, and nuclear accumulation of GRK5, which depended on its phosphatase activity. Finally, using siRNA against GRK5, we found that GRK5 was necessary for the hypertrophic response induced by PHLPP2 knockdown. Our findings demonstrate for the first time a novel regulation of GRK5 by the phosphatase PHLPP2, which modulates hypertrophic growth. Understanding the signaling pathways affected by PHLPP2 has potential for new therapeutic targets in the treatment of cardiac hypertrophy and failure., (© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.)

G protein-coupled receptor (GPCR) kinases (GRKs) regulate the desensitization and internalization of GPCRs. Two of these, GRK2 and GRK5, are upregulated in heart failure and are promising targets for heart failure treatment. Although there have been several reports of potent and selective inhibitors of GRK2 there are few for GRK5. Herein, we describe a ligand docking approach utilizing the crystal structures of the GRK2-Gβγ·GSK180736A and GRK5·CCG215022 complexes to search for amide substituents predicted to confer GRK2 and/or GRK5 potency and selectivity. From this campaign, we successfully generated two new potent GRK5 inhibitors, although neither exhibited selectivity over GRK2., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

We have recently demonstrated that the amino-terminal domain of G protein coupled receptor kinase (GRK) type 5, (GRK5-NT) inhibits NFκB activity in cardiac cells leading to a significant amelioration of LVH. Since GRK5-NT is known to bind calmodulin, this study aimed to evaluate the functional role of GRK5-NT in the regulation of calcium-calmodulin-dependent transcription factors. We found that the overexpression of GRK5-NT in cardiomyoblasts significantly reduced the activation and the nuclear translocation of NFAT and its cofactor GATA-4 in response to phenylephrine (PE). These results were confirmed in vivo in spontaneously hypertensive rats (SHR), in which intramyocardial adenovirus-mediated gene transfer of GRK5-NT reduced both wall thickness and ventricular mass by modulating NFAT and GATA-4 activity. To further verify in vitro the contribution of calmodulin in linking GRK5-NT to the NFAT/GATA-4 pathway, we examined the effects of a mutant of GRK5 (GRK5-NTPB), which is not able to bind calmodulin. When compared to GRK5-NT, GRK5-NTPB did not modify PE-induced NFAT and GATA-4 activation. In conclusion, this study identifies a double effect of GRK5-NT in the inhibition of LVH that is based on the regulation of multiple transcription factors through means of different mechanisms and proposes the amino-terminal sequence of GRK5 as a useful prototype for therapeutic purposes., Competing Interests: The Authors declare no conflicts of interest.

Estradiol (E 2 ) prevents cardiac hypertrophy, and these protective actions are mediated by estrogen receptor (ER)α and ERβ. The G protein-coupled estrogen receptor (GPER) mediates many estrogenic effects, and its activation in the heart has been observed in ischemia and reperfusion injury or hypertension models; however, the underlying mechanisms need to be fully elucidated. Herein, we investigated whether the protective effect of E 2 against cardiomyocyte hypertrophy induced by endothelin-1 (ET-1) is mediated by GPER and the signaling pathways involved. Isolated neonatal female rat cardiomyocytes were treated with ET-1 (100 nmol/l) for 48 h in the presence or absence of E 2 (10 nmol/l) or GPER agonist G-1 (10 nmol/l) and GPER antagonist G-15 (10 nmol/l). ET-1 increased the surface area of cardiomyocytes, and this was associated with increased expression of atrial and brain natriuretic peptides. Additionally, ET-1 increased the phosphorylation of extracellular signal-related protein kinases-1/2 (ERK1/2). Notably, E 2 or G-1 abolished the hypertrophic actions of ET-1, and that was reversed by G-15. Likewise, E 2 reversed the ET-1-mediated increase of ERK1/2 phosphorylation as well as the decrease of phosphorylated Akt and its upstream activator 3-phosphoinositide-dependent protein kinase-1 (PDK1). These effects were inhibited by G-15, indicating that they are GPER dependent. Confirming the participation of GPER, siRNA silencing of GPER inhibited the antihypertrophic effect of E 2 . In conclusion, E 2 plays a key role in antagonizing ET-1-induced hypertrophy in cultured neonatal cardiomyocytes through GPER signaling by a mechanism involving activation of the PDK1 pathway, which would prevent the increase of ERK1/2 activity and consequently the development of hypertrophy.

Lung cancer is the leading cause of cancer-related deaths worldwide, and non-small-cell lung cancer (NSCLC) accounts for about 80% of all cases, which is the major subgroup of lung cancer. G protein-coupled receptor kinase 5 (GRK5) has been demonstrated to play pivotal roles in both development and progression of several pathological conditions including cancer. Here, we found that GRK5 expression was significantly increased in 539 NSCLC cancerous tissues than that in 99 normal non-cancerous tissues by immunohistochemistry analysis; we also showed intensive higher positive staining percentage in female and adenocarcinoma (ADC) NSCLC patients than that in male and squamous cell carcinoma (SCC) patients, respectively. In addition, GRK5 high expression NSCLC patients had a worse overall survival rate than the low expression patients. We provided evidence showing that both the mRNA and protein expression levels of GRK5 were increased in NSCLC cancerous cell lines (GLC-82, SPC-A-1, H520, H838, H358, A549, and H1299) comparing with that in normal human bronchial epithelium cell line (BEAS-2B), and identified many GRK5 mutations in NSCLC cancerous tissues. In addition, we found that depletion of GRK5 inhibited NSCLC cancerous cell proliferation, migration in vitro, and xenograft tumor formation in vivo. Furthermore, GRK5 knockdown promoted cell cycle arrest at G2/M phase and induced cellular apoptosis. In summary, our data reveal an oncogenic role of GRK5 in NSCLC progression, indicating that GRK5 could be used as a new therapeutic target in future.

Opioids are powerful analgesics, but also carry significant side effects and abuse potential. Here we describe a modulator of the μ-opioid receptor (MOR1), the transient receptor potential channel subfamily vanilloid member 1 (TRPV1). We show that TRPV1 binds MOR1 and blocks opioid-dependent phosphorylation of MOR1 while leaving G protein signaling intact. Phosphorylation of MOR1 initiates recruitment and activation of the β-arrestin pathway, which is responsible for numerous opioid-induced adverse effects, including the development of tolerance and respiratory depression. Phosphorylation stands in contrast to G protein signaling, which is responsible for the analgesic effect of opioids. Calcium influx through TRPV1 causes a calcium/calmodulin-dependent translocation of G protein-coupled receptor kinase 5 (GRK5) away from the plasma membrane, thereby blocking its ability to phosphorylate MOR1. Using TRPV1 to block phosphorylation of MOR1 without affecting G protein signaling is a potential strategy to improve the therapeutic profile of opioids., Competing Interests: The authors declare no conflict of interest.

Objective: Acute seizures induced dendritic formation and synaptogenesis promotes aberrant circuitry development and further aggravates underlying conditions towards chronic epilepsy. The G protein-coupled receptor kinase-5 (GRK5) served as a key modulator in neurogenesis and the establishment of functional neuronal circuitry. This included dendritic development, as its dysfunction could cause different central nervous system disorders, including Alzheimer's disease. However, the involvement of GRK5 in the progression of epilepsy remains unclear. The purpose of this study is to investigate the involvement of GRK5 in epilepsy, as well as its potential correlation with dendritic formation after status epilepticus., Methods: 120 rats were divided into control and model groups. The rats in the model group were injected intraperitoneally with lithium chloride-pilocarpine hydrochloride to establish the rat model of status epilepticus (SE). The brain and hippocampus were collected at 1, 3, 7, 14 and 28days post SE induction. The expression and distribution of GRK5 and the dendritic marker microtubule-associated protein-2 (MAP-2) were detected in the hippocampus via western blot or immunohistochemistry. The co-localization of GRK5 with MAP-2 was examined via laser confocal double immunofluorescence staining. The interactions between GRK5 and MAP-2 during epileptogenesis were evaluated via immunoprecipitation., Results: GRK5 was distributed in all areas of the hippocampus. Its expression was significantly up-regulated in the hippocampal CA1, DG, and H areas at 7d and 14d after SE. After 14d it began to reduce. and then reduced. MAP-2 primarily existed in the neuronal dendrites of the hippocampal subregion. Its expression was enhanced at 3d. It reached its maximum level at 14d after SE, where it then began to fall. The confocal microscope analysis revealed that GRK5 was co-located well within MAP-2 positive cells. The interaction between GRK5 and MAP-2 became enhanced at 7d and 14d after SE., Conclusions: GRK5 was involved in the development of epilepsy. It was associated with dendritic formation in epilepsy. This study provides a new perspective for elucidating the epilepsy pathogenesis. The concrete mechanisms of the GRK5 within epileptogenesis require further research., (Copyright © 2017 Elsevier B.V. All rights reserved.)

G protein-coupled receptors (GPCRs) are essential for transferring extracellular signals into carefully choreographed intracellular responses controlling diverse aspects of cell physiology. The duration of GPCR-mediated signaling is primarily regulated via GPCR kinase (GRK)-mediated phosphorylation of activated receptors. Although many GRK structures have been reported, the mechanisms underlying GRK activation are not well-understood, in part because it is unknown how these structures map to the conformational landscape available to this enzyme family. Unlike most other AGC kinases, GRKs rely on their interaction with GPCRs for activation and not phosphorylation. Here, we used principal component analysis of available GRK and protein kinase A crystal structures to identify their dominant domain motions and to provide a framework that helps evaluate how close each GRK structure is to being a catalytically competent state. Our results indicated that disruption of an interface formed between the large lobe of the kinase domain and the regulator of G protein signaling homology domain (RHD) is highly correlated with establishment of the active conformation. By introducing point mutations in the GRK5 RHD-kinase domain interface, we show with both in silico and in vitro experiments that perturbation of this interface leads to higher phosphorylation activity. Navigation of the conformational landscape defined by this bioinformatics-based study is likely common to all GPCR-activated GRKs., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

The phosphorylation of agonist-occupied G-protein-coupled receptors (GPCRs) by GPCR kinases (GRKs) functions to turn off G-protein signaling and turn on arrestin-mediated signaling. While a structural understanding of GPCR/G-protein and GPCR/arrestin complexes has emerged in recent years, the molecular architecture of a GPCR/GRK complex remains poorly defined. We used a comprehensive integrated approach of cross-linking, hydrogen-deuterium exchange mass spectrometry (MS), electron microscopy, mutagenesis, molecular dynamics simulations, and computational docking to analyze GRK5 interaction with the β 2 -adrenergic receptor (β 2 AR). These studies revealed a dynamic mechanism of complex formation that involves large conformational changes in the GRK5 RH/catalytic domain interface upon receptor binding. These changes facilitate contacts between intracellular loops 2 and 3 and the C terminus of the β 2 AR with the GRK5 RH bundle subdomain, membrane-binding surface, and kinase catalytic cleft, respectively. These studies significantly contribute to our understanding of the mechanism by which GRKs regulate the function of activated GPCRs. PAPERCLIP., (Copyright © 2017 Elsevier Inc. All rights reserved.)

It is well known that Src tyrosine kinase, insulin-like growth factor 1 receptor (IGF-IR), and focal adhesion kinase (FAK) play important roles in prostate cancer (PrCa) development and progression. Src, which signals through FAK in response to integrin activation, has been implicated in many aspects of tumor biology, such as cell proliferation, metastasis, and angiogenesis. Furthermore, Src signaling is known to crosstalk with IGF-IR, which also promotes angiogenesis. In this study, we demonstrate that c-Src, IGF-IR, and FAK are packaged into exosomes (Exo), c-Src in particular being highly enriched in Exo from the androgen receptor (AR)-positive cell line C4-2B and AR-negative cell lines PC3 and DU145. Furthermore, we show that the active phosphorylated form of Src (Src pY416 ) is co-expressed in Exo with phosphorylated FAK (FAK pY861 ), a known target site of Src, which enhances proliferation and migration. We further demonstrate for the first time exosomal enrichment of G-protein-coupled receptor kinase (GRK) 5 and GRK6, both of which regulate Src and IGF-IR signaling and have been implicated in cancer. Finally, Src pY416 and c-Src are both expressed in Exo isolated from the plasma of prostate tumor-bearing TRAMP mice, and those same mice have higher levels of exosomal c-Src than their wild-type counterparts. In summary, we provide new evidence that active signaling molecules relevant to PrCa are enriched in Exo, and this suggests that the Src signaling network may provide useful biomarkers detectable by liquid biopsy, and may contribute to PrCa progression via Exo. J. Cell. Biochem. 118: 66-73, 2017. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

Urotensin II (UII) is a neural hormone that induces cardiac hypertrophy and may be involved in the pathogenesis of cardiac remodeling and heart failure. Hypertrophy has been linked to histone deacetylase 5 (HDAC5) phosphorylation and nuclear factor κB (NF-κB) translocation, both of which are predominantly mediated by G protein-coupled receptor kinase 5 (GRK5). In the present study, we found that UII rapidly and strongly stimulated nuclear export of HDAC5 and nuclear import of NF-κB in H9c2 cells overexpressing the urotensin II receptor (H9c2 UT ). Hence, we hypothesized that GRK5 and its signaling pathway may play a role in UII-mediated cellular hypertrophy. H9c2 UT cells were transduced with a GRK5 small hairpin RNA interference recombinant lentivirus, resulting in the down-regulation of GRK5. Under UII stimulation, reduced levels of GRK5 in H9c2 UT cells led to suppression of UII-mediated HDAC5 phosphorylation and activation of the NF-κB signaling pathway. In contrast, UII-mediated activations of ERK1/2 and GSK3α/β were not affected by down-regulation of GRK5. In a cellular hypertrophy assay, down-regulation of GRK5 significantly suppressed UII-mediated hypertrophy of H9c2 UT cells. Furthermore, UII-mediated cellular hypertrophy was inhibited by amlexanox, a selective GRK5 inhibitor, in H9c2 UT cells and neonatal cardiomyocytes. Our results suggest that GRK5 may be involved in a UII-mediated hypertrophic response via activation of NF-κB and HDAC5 at least in part by ERK1/2 and GSK3α/β-independent pathways.

Activation of protease-activated receptor 1 (PAR1) by activated protein C (APC) and thrombin elicits paradoxical cytoprotective and cytotoxic signaling responses in vascular endothelial cells through cleavage of the receptor at Arg-46 and Arg-41 protease recognition sites, respectively. It has been reported that unlike a disruptive G-protein-mediated PAR1 signaling by thrombin, APC induces a protective β-arrestin-2 biased PAR1 signaling by unknown mechanisms. We hypothesize that the occupancy of endothelial protein C receptor (EPCR) by the Gla-domain of protein C/APC is responsible for the β-arrestin-2 biased PAR1 signaling independent of the protease cleavage site. To test this hypothesis, we monitored the signaling specificity of thrombin in endothelial cells in response to lipopolysaccharide (LPS) with or without pretreatment of cells with protein C-S195A. The PAR1-dependent recruitment of β-arrestin-2 in response to LPS by both APC and thrombin was analyzed by functional, gene silencing, and signaling assays. Results indicate that similar to APC, thrombin exerts cytoprotective effects via β-arrestin-2 biased PAR1 signaling. Similar to APC, thrombin triggered β-arrestin-2-dependent recruitment of disheveled 2 (Dvl-2) in PC-S195A pretreated cells. Further studies in HeLa cells transfected with PAR1 constructs revealed that EPCR occupancy initiates β-arrestin-2 biased PAR1 signaling independent of the protease cleavage sites. We demonstrate that EPCR occupancy recruits G-protein coupled receptor kinase 5, thereby inducing β-arrestin-2 biased PAR1 signaling by both APC and thrombin. In support of a physiological relevance for these results, intraperitoneal administration of PC-S195A conferred a cytoprotective effect for thrombin in an in vivo inflammatory model., (© 2016 by The American Society of Hematology.)

Heart failure (HF) is a global epidemic with the highest degree of mortality and morbidity of any disease presently studied. G protein-coupled receptors (GPCRs) are prominent regulators of cardiovascular function. Activated GPCRs are "turned off" by GPCR kinases (GRKs) in a process known as "desensitization". GRKs 2 and 5 are highly expressed in the heart, and known to be upregulated in HF. Over the last 20 years, both GRK2 and GRK5 have been demonstrated to be critical mediators of the molecular alterations that occur in the failing heart. In the present review, we will highlight recent findings that further characterize "non-canonical" GRK signaling observed in HF. Further, we will also present potential therapeutic strategies (i.e. small molecule inhibition, microRNAs, gene therapy) that may have potential in combating the deleterious effects of GRKs in HF., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Agonist-induced internalization plays a key role in the tight regulation of the extent and duration of G protein-coupled receptor signaling. Previously, we have shown that the Bombyx corazonin receptor (BmCrzR) activates both Gαq- and Gαs-dependent signaling cascades. However, the molecular mechanisms involved in the regulation of the internalization and desensitization of BmCrzR remain to be elucidated. Here, vectors for expressing BmCrzR fused with enhanced green fluorescent protein (EGFP) at the C-terminal end were used to further characterize BmCrzR internalization. We found that the BmCrzR heterologously expressed in HEK-293 and BmN cells was rapidly internalized from the plasma membrane into the cytoplasm in a concentration- and time-dependent manner via a β-arrestin (Kurtz)-dependent and clathrin-independent pathway in response to agonist challenge. While most of the internalized receptors were recycled to the cell surface via early endosomes, some others were transported to lysosomes for degradation. Assays using RNA interference revealed that both GRK2 and GRK5 were essentially involved in the regulation of BmCrzR phosphorylation and internalization. Further investigations indicated that the identified cluster of Ser/Thr residues ((411)TSS(413)) was responsible for GRK-mediated phosphorylation and internalization. This is the first detailed investigation of the internalization and trafficking of Bombyx corazonin receptors.

G protein-coupled receptor kinase 5 (GRK5) is a serine/threonine kinase previously shown to mediate polymicrobial sepsis-induced inflammation. The goal of the present study was to examine the role of GRK5 in monomicrobial pulmonary infection by using an intratracheal Escherichia coli infection model of pneumonia. We used sublethal and lethal doses of E. coli to examine the mechanistic differences between low-grade and high-grade inflammation induced by E. coli infection. With a sublethal dose of E. coli, GRK5 knockout (KO) mice exhibited higher plasma CXCL1/KC levels and enhanced lung neutrophil recruitment early after infection, and lower bacterial loads, than wild-type (WT) mice. The inflammatory response was also diminished, and resolution of inflammation advanced, in the lungs of GRK5 KO mice. In contrast to the reduced bacterial loads in GRK5 KO mice following a sublethal dose, at a lethal dose of E. coli, the bacterial burdens remained high in GRK5 KO mice relative to those in WT mice. This occurred in spite of enhanced plasma CXCL1 levels as well as neutrophil recruitment in the KO mice. But the recruited neutrophils (following high-dose infection) exhibited decreased CD11b expression and reduced reactive oxygen species production, suggesting decreased neutrophil activation or increased neutrophil exhaustion in the GRK5 KO mice. In agreement with the increased bacterial burden, KO mice showed poorer survival than WT mice following E. coli infection at a lethal dose. Overall, our data suggest that GRK5 negatively regulates CXCL1/KC levels during bacterial pneumonia but that the role of GRK5 in the clinical outcome in this model is dependent on the bacterial dose., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

As the average world-wide lifespan continues to increase, heart failure (HF) has dramatically increased in incidence leading to the highest degree of mortality and morbidity of any disease presently studied. G protein-coupled receptors (GPCRs) play a prominent role in regulation of cardiovascular function. GPCRs are effectively "turned off" by GPCR kinases (GRKs) in a process known as "desensitization". GRKs 2 and 5 are highly expressed in the heart, and known to be upregulated in HF. Over the last 20years, the role of GRK2 in HF has been widely studied. However, until recently, the role of GRK5 in cardiac pathophysiology had yet to be elucidated. In the present review, we will focus on GRK5's role in the myocardium in normal physiology, and its apparent critical role in the progression of HF. Further, we will also present potential therapeutic strategies (i.e. small molecule inhibition, gene therapy) that may have potential in combating the deleterious effects of GRK5 in HF., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Elevated sympathetic nervous system (SNS) activity aggravates several diseases, including heart failure. The molecular cause(s) underlying this SNS hyperactivity are not known. We have previously uncovered a neurohormonal mechanism, operating in adrenomedullary chromaffin cells, by which circulating catecholamine (CA) levels increase in heart failure: severe dysfunction of the adrenal α2-adrenergic receptors (ARs) due to the up-regulation of G protein-coupled receptor-kinase (GRK)-2, the kinase that desensitizes them. Herein we looked at the potential signaling mechanisms that bring about this GRK2 elevation in chromaffin cells. We found that chronic CA treatment of either PC12 or rat primary chromaffin cells can in itself result in GRK2 transcriptional up-regulation through α2ARs-Gi/o proteins-Src-ERK1/2. The resultant GRK2 increase severely enhances the α2AR desensitization/down-regulation elevating not only CA release but also CA biosynthesis, as evidenced by tyrosine hydroxylase up-regulation. Finally, GRK2 knockdown leads to enhanced apoptosis of PC12 cells, indicating an essential role for GRK2 in chromaffin cell homeostasis/survival. In conclusion, chromaffin cell GRK2 mediates a positive feedback loop that feeds into CA secretion, thereby enabling the adrenomedullary component of the SNS to turn itself on.

Receptor activity-modifying protein 2 (RAMP2) enables the calcitonin receptor-like receptor (CLR, a family B GPCR) to form the type 1 adrenomedullin receptor (AM1 receptor). Here, we investigated the effects of the five non-visual GPCR kinases (GRKs 2 through 6) on the cell surface expression of the human (h)AM1 receptor by cotransfecting each of these GRKs into HEK-293 cells that stably expressed hRAMP2. Flow cytometric analysis revealed that when coexpressed with GRK4 or GRK5, the cell surface expression of the AM1 receptor was markedly decreased prior to stimulation with AM, thereby attenuating both the specific [(125)I]AM binding and AM-induced cAMP production. These inhibitory effects of both GRKs were abolished by the replacement of the cytoplasmic C-terminal tail (C-tail) of CLR with that of the calcitonin receptor (a family B GPCR) or β2-adrenergic receptor (a family A GPCR). Among the sequentially truncated CLR C-tail mutants, those lacking the five residues 449-453 (Ser-Phe-Ser-Asn-Ser) abolished the inhibition of the cell surface expression of CLR via the overexpression of GRK4 or GRK5. Thus, we provided new insight into the function of GRKs in agonist-unstimulated GPCR trafficking using a recombinant AM1 receptor and further determined the region of the CLR C-tail responsible for this GRK function., (Copyright © 2016 Elsevier Inc. All rights reserved.)

To explore if genic expression of β(1)- or β(2)-adrenoceptors (ARs) exhibits a common regulatory pattern with G protein-coupled receptor kinase (GRK) 2, GRK3, or GRK5 expression, we determined messenger RNA levels for these genes in different tissues from human and animal models of cardiovascular disease. We measured genic expression by qRT polymerase chain reaction in the left and right ventricles or peripheral blood mononuclear cells from healthy (n = 21), hypertensive (n = 20), heart failure (n = 24), and heart transplanted patients (n = 17) or in left ventricle, peripheral blood mononuclear cells, and kidney from spontaneously hypertensive rats or L-N-methyl-arginine-induced hypertensive rats and their respective controls (n = 4-5). In diseased versus healthy subjects and rats, parallel changes in messenger RNA levels of GRK2 and β(2)-AR or GRK5 and β(1)-AR were observed in each territory. Therefore, without excluding other regulatory mechanisms, the parallelism observed suggests a common regulatory pattern for the β(1)-AR/GRK5 and β(2)-AR/GRK2 genes, which is independent of cellular type or pathology. This highlights the need to focus not only on GRKs but also on β(1)- or β(2)-AR changes to completely understand the involvement of β-AR/GRK pathways in cardiovascular diseases.

G protein-coupled receptor kinases (GRKs) play an important role in the desensitization of G protein-mediated signaling of G protein-coupled receptors (GPCRs). The level of interest in mapping their phosphorylation sites has increased because recent studies suggest that the differential pattern of receptor phosphorylation has distinct biological consequences. In vitro phosphorylation experiments using well-controlled systems are useful for deciphering the complexity of these physiological reactions and understanding the targeted event. Here, we report on the phosphorylation of the class A GPCR neurotensin receptor 1 (NTSR1) by GRKs under defined experimental conditions afforded by nanodisc technology. Phosphorylation of NTSR1 by GRK2 was agonist-dependent, whereas phosphorylation by GRK5 occurred in an activation-independent manner. In addition, the negatively charged lipids in the immediate vicinity of NTSR1 directly affect phosphorylation by GRKs. Identification of phosphorylation sites in agonist-activated NTSR1 revealed that GRK2 and GRK5 target different residues located on the intracellular receptor elements. GRK2 phosphorylates only the C-terminal Ser residues, whereas GRK5 phosphorylates Ser and Thr residues located in intracellular loop 3 and the C-terminus. Interestingly, phosphorylation assays using a series of NTSR1 mutants show that GRK2 does not require acidic residues upstream of the phospho-acceptors for site-specific phosphorylation, in contrast to the β2-adrenergic and μ-opioid receptors. Differential phosphorylation of GPCRs by GRKs is thought to encode a particular signaling outcome, and our in vitro study revealed NTSR1 differential phosphorylation by GRK2 and GRK5.