Neuronal GRK2 regulates microglial activation and contributes to electroacupuncture analgesia on inflammatory pain in mice.

BACKGROUND: G protein coupled receptor kinase 2 (GRK2) has been demonstrated to play a crucial role in the development of chronic pain. Acupuncture is an alternative therapy widely used for pain management. In this study, we investigated the role of spinal neuronal GRK2 in electroacupuncture (EA) analgesia. METHODS: The mice model of inflammatory pain was built by subcutaneous injection of Complete Freund's Adjuvant (CFA) into the plantar surface of the hind paws. The mechanical allodynia of mice was examined by von Frey test. The mice were subjected to EA treatment (BL60 and ST36 acupuncture points) for 1 week. Overexpression and downregulation of spinal neuronal GRK2 were achieved by intraspinal injection of adeno associated virus (AAV) containing neuron-specific promoters, and microglial activation and neuroinflammation were evaluated by real-time PCR. RESULTS: Intraplantar injection with CFA in mice induced the decrease of GRK2 and microglial activation along with neuroinflammation in spinal cord. EA treatment increased the spinal GRK2, reduced neuroinflammation, and significantly decreased CFA-induced mechanical allodynia. The effects of EA were markedly weakened by non-cell-specific downregulation of spinal GRK2. Further, intraspinal injection of AAV containing neuron-specific promoters specifically downregulated neuronal GRK2, and weakened the regulatory effect of EA on CFA-induced mechanical allodynia and microglial activation. Meanwhile, overexpression of spinal neuronal GRK2 decreased mechanical allodynia. All these indicated that the neuronal GRK2 mediated microglial activation and neuroinflammation, and subsequently contributed to CFA-induced inflammatory pain. CONCLUSION: The restoration of the spinal GRK2 and subsequent suppression of microglial activation and neuroinflammation might be an important mechanism for EA analgesia. Our findings further suggested that the spinal GRK2, especially neuronal GRK2, might be the potential target for EA analgesia and pain management, and we provided a new experimental basis for the EA treatment of pain.

Cell therapy rescues aging-induced beta-1 adrenergic receptor and GRK2 dysfunction in the coronary microcirculation.

Our past study showed that coronary arterioles isolated from adipose-derived stromal vascular fraction (SVF)-treated rats showed amelioration of the age-related decrease in vasodilation to beta-adrenergic receptor (ß-AR) agonist and improved ß-AR-dependent coronary flow and microvascular function in a model of advanced age. We hypothesized that intravenously (i.v.) injected SVF improves coronary microvascular function in aged rats by re-establishing the equilibrium of the negative regulators of the internal adrenergic signaling cascade, G-protein receptor kinase 2 (GRK2) and G-alpha inhibitory (Gαi) proteins, back to youthful levels. Female Fischer-344 rats aged young (3 months, n = 24), old (24 months, n = 26), and old animals that received 1 × 107 green fluorescent protein (GFP+) SVF cells (O + SVF, n = 11) 4 weeks prior to sacrifice were utilized. Overnight urine was collected prior to sacrifice for catecholamine measurements. Cardiac samples were used for western blotting while coronary arterioles were isolated for pressure myography studies, immunofluorescence staining, and RNA sequencing. Coronary microvascular levels of the ß1 adrenergic receptor are decreased with advancing age, but this decreased expression was rescued by SVF treatment. Aging led to a decrease in phosphorylated GRK2 in cardiomyocytes vs. young control with restoration of phosphorylation status by SVF. In vessels, there was no change in genetic transcription (RNAseq) or protein expression (immunofluorescence); however, inhibition of GRK2 (paroxetine) led to improved vasodilation to norepinephrine in the old control (OC) and O + SVF, indicating greater GRK2 functional inhibition of ß1-AR in aging. SVF works to improve adrenergic-mediated vasodilation by restoring the ß1-AR population and mitigating signal cascade inhibitors to improve vasodilation.

Neuronal GRK2 regulates microglial activation and contributes to electroacupuncture analgesia on inflammatory pain in mice

BACKGROUND: G protein coupled receptor kinase 2 (GRK2) has been demonstrated to play a crucial role in the development of chronic pain. Acupuncture is an alternative therapy widely used for pain management. In this study, we investigated the role of spinal neuronal GRK2 in electroacupuncture (EA) analgesia. METHODS: The mice model of inflammatory pain was built by subcutaneous injection of Complete Freund's Adjuvant (CFA) into the plantar surface of the hind paws. The mechanical allodynia of mice was examined by von Frey test. The mice were subjected to EA treatment (BL60 and ST36 acupuncture points) for 1 week. Overexpression and down-regulation of spinal neuronal GRK2 were achieved by intraspinal injection of adeno associated virus (AAV) containing neuron-specific promoters, and microglial activation and neuroinflammation were evaluated by real-time PCR. RESULTS: Intraplantar injection with CFA in mice induced the decrease of GRK2 and microglial activation along with neuroinflammation in spinal cord. EA treatment increased the spinal GRK2, reduced neuroinflammation, and significantly decreased CFA-induced mechanical allodynia. The effects of EA were markedly weakened by non-cell-specific downregulation of spinal GRK2. Further, intraspinal injection of AAV containing neuron-specific promoters specifically downregulated neuronal GRK2, and weakened the regulatory effect of EA on CFA-induced mechanical allodynia and microglial activation. Meanwhile, overexpression of spinal neuronal GRK2 decreased mechanical allodynia. All these indicated that the neuronal GRK2 mediated microglial activation and neuroinflammation, and subsequently contributed to CFA-induced inflammatory pain. CONCLUSION: The restoration of the spinal GRK2 and subsequent suppression of microglial activation and neuroinflammation might be an important mechanism for EA analgesia. Our findings further suggested that the spinal GRK2, especially neuronal GRK2, might be the potential target for EA analgesia and pain management, and we provided a new experimental basis for the EA treatment of pain.

Dissecting the roles of GRK2 and GRK3 in µ-opioid receptor internalization and ß-arrestin2 recruitment using CRISPR/Cas9-edited HEK293 cells.

Most G protein-coupled receptors (GPCRs) recruit ß-arrestins and internalize upon agonist stimulation. For the µ-opioid receptor (µ-OR), this process has been linked to development of opioid tolerance. GPCR kinases (GRKs), particularly GRK2 and GRK3, have been shown to be important for µ-OR recruitment of ß-arrestin and internalization. However, the contribution of GRK2 and GRK3 to ß-arrestin recruitment and receptor internalization, remain to be determined in their complete absence. Using CRISPR/Cas9-mediated genome editing we established HEK293 cells with knockout of GRK2, GRK3 or both to dissect their individual contributions in ß-arrestin2 recruitment and µ-OR internalization upon stimulation with four different agonists. We showed that GRK2/3 removal reduced agonist-induced µ-OR internalization and ß-arrestin2 recruitment substantially and we found GRK2 to be more important for these processes than GRK3. Furthermore, we observed a sustained and GRK2/3 independent component of ß-arrestin2 recruitment to the plasma membrane upon µ-OR activation. Rescue expression experiments restored GRK2/3 functions. Inhibition of GRK2/3 using the small molecule inhibitor CMPD101 showed a high similarity between the genetic and pharmacological approaches, cross-validating the specificity of both. However, off-target effects were observed at high CMPD101 concentrations. These GRK2/3 KO cell lines should prove useful for a wide range of studies on GPCR function.

Agonist-selective recruitment of engineered protein probes and of GRK2 by opioid receptors in living cells.

G protein-coupled receptors (GPCRs) signal through allostery, and it is increasingly clear that chemically distinct agonists can produce different receptor-based effects. It has been proposed that agonists selectively promote receptors to recruit one cellular interacting partner over another, introducing allosteric 'bias' into the signaling system. However, the underlying hypothesis - that different agonists drive GPCRs to engage different cytoplasmic proteins in living cells - remains untested due to the complexity of readouts through which receptor-proximal interactions are typically inferred. We describe a cell-based assay to overcome this challenge, based on GPCR-interacting biosensors that are disconnected from endogenous transduction mechanisms. Focusing on opioid receptors, we directly demonstrate differences between biosensor recruitment produced by chemically distinct opioid ligands in living cells. We then show that selective recruitment applies to GRK2, a biologically relevant GPCR regulator, through discrete interactions of GRK2 with receptors or with G protein beta-gamma subunits which are differentially promoted by agonists.

About a third of all drugs work by targeting a group of proteins known as G-protein coupled receptors, or GPCRs for short. These receptors are found on the surface of cells and transmit messages across the cell's outer barrier. When a signaling molecule, like a hormone, is released in the body, it binds to a GPCR and changes the receptor's shape. The change in structure affects how the GPCR interacts and binds to other proteins on the inside of the cell, triggering a series of reactions that alter the cell's activity. Scientists have previously seen that a GPCR can trigger different responses depending on which signaling molecule is binding on the surface of the cell. However, the mechanism for this is unknown. One hypothesis is that different signaling molecules change the GPCR's preference for binding to different proteins on the inside of the cell. The challenge has been to observe this happening without interfering with the process. Stoeber et al. have now tested this idea by attaching fluorescent tags to proteins that bind to activated GPCRs directly and without binding other signaling proteins. This meant these proteins could be tracked under a microscope as they made their way to bind to the GPCRs. Stoeber et al. focused on one particular GPCR, known as the opioid receptor, and tested the binding of two different opioid signaling molecules, etorphine and Dynorphin A. The experiments revealed that the different opioids did affect which of the engineered proteins would preferentially bind to the opioid receptor. This was followed by a similar experiment, where the engineered proteins were replaced with another protein called GRK2, which binds to the opioid receptor under normal conditions in the cell. This showed that GRK2 binds much more strongly to the opioid receptor when Dynorphin A is added compared to adding etorphine. These findings show that GPCRs can not only communicate that a signaling molecule is binding but can respond differently to convey what molecule it is more specifically. This could be important in developing drugs, particularly to specifically trigger the desired response and reduce side effects. Stoeber et al. suggest that an important next step for research is to understand how the GPCRs preferentially bind to different proteins.

Biased S1PR1 Signaling in B Cells Subverts Responses to Homeostatic Chemokines, Severely Disorganizing Lymphoid Organ Architecture.

Ligand-engaged chemoattractant receptors trigger Gαi subunit nucleotide exchange, stimulating the activation of downstream effector molecules. Activated chemoattractant receptors also dock G protein-coupled receptor kinases (GRKs) that help mediate receptor desensitization. In this study, we show that the B cell-specific loss of GRK2 severely disrupts B cell trafficking and immune cell homeostasis. The GRK2 deficiency in developing murine B cells leads to a severe immune phenotype, including a major reduction of bone marrow IgD+ cells, splenomegaly with a loss of white pulp and grossly expanded red pulp, a deficit of Peyer patches, and small lymph nodes with marked reductions in B cell numbers. The major phenotypes in these mice arise from excessive S1PR1 signaling combined with inadequate homeostatic chemokine receptor signaling. CXCL13 signaling is the most severely compromised. In B cells, our data also indicate that S1PR1 signals constitutively, as blocking S1PR1 signaling with an S1PR1 antagonist enhanced CXCL13-triggered wild-type B cell migration. Furthermore, blocking S1PR1 signaling in the GRK2-deficient B cells partially corrected their poor response to chemokines. Treating mice lacking GRK2 expression in their B cells with an S1PR1 antagonist partially normalized B cell trafficking into lymph node and splenic follicles. These findings reveal the critical interdependence of Gαi-linked signaling pathways in controlling B lymphocyte trafficking.

Carvedilol protects against hepatic ischemia/reperfusion injury in high-fructose/high-fat diet-fed mice: Role of G protein-coupled receptor kinase 2 and 5.

Hepatic ischemia/reperfusion injury (H-IRI) is associated with irreversible liver damage. The current study aimed to investigate the protective effect of carvedilol against H-IRI in high-fructose high-fat diet (HFrHFD)-fed mice and the role of G protein-coupled receptor kinase 2 and 5 (GRK2 and GRK5). Mice were fed HFrHFD for 16 weeks; then mice were subjected to 30 min of ischemia followed by 1 h of reperfusion at the end of feeding period. Carvedilol (20 mg/kg, i.p.) was administered 30 min before ischemia. To explore the role of GRK2 and GRK5 in mediating carvedilol effects, paroxetine (GRK2 inhibitor, 10 mg/kg, i.p.) and amlexanox (GRK5 inhibitor, 25 mg/kg, i.p.) were administered 30 min before carvedilol administration. Liver function, histopathology and hepatic oxidative stress, as well as inflammatory and apoptotic markers were measured at the end of the experiment. In addition, adrenergic receptor downstream signals were measured in the liver. Results showed increased markers of liver injury (ALT and AST) in mice subjected to H-IRI. Moreover, liver injury was associated with slight collagen deposits as revealed by histopathology and elevated hepatic levels of oxidative stress, inflammatory and apoptotic markers. On the other hand, carvedilol protected mice against H-IRI and improved all associated pathological changes. Furthermore, pre-injection of either GRK2 or GRK5 inhibitor did not change carvedilol effects on serum ALT level and liver collagen deposits, while increased its antioxidant, anti-inflammatory and anti-apoptotic effects. In conclusion, carvedilol protects against H-IRI in HFrHFD-fed mice. GRK2 and GRK5 may not play a potential role in mediating this effect.

Differential regulation of ß2-adrenoceptor and adenosine A2B receptor signalling by GRK and arrestin proteins in arterial smooth muscle.

Generation of cAMP through Gs-coupled G protein-coupled receptor (GPCR) [e.g. ß2-adrenoceptor (ß2AR), adenosine A2B receptor (A2BR)] activation, induces arterial smooth muscle relaxation, counteracting the actions of vasoconstrictors. Gs-coupled GPCR signalling is regulated by G protein-coupled receptor kinases (GRK) and arrestin proteins, and dysregulation of Gs/GPCR signalling is thought play a role in the development of hypertension, which may be a consequence of enhanced GRK2 and/or arrestin expression. However, despite numerous studies indicating that ß2AR and A2BR can be substrates for GRK/arrestin proteins, currently little is known regarding GRK/arrestin regulation of these endogenous receptors in arterial smooth muscle. Here, endogenous GRK isoenzymes and arrestin proteins were selectively depleted using RNA-interference in rat arterial smooth muscle cells (RASM) and the consequences of this for ß2AR- and A2BR-mediated adenylyl cyclase (AC) signalling were determined by assessing cAMP accumulation. GRK2 or GRK5 depletion enhanced and prolonged ß2AR/AC signalling, while combined deletion of GRK2/5 has an additive effect. Conversely, activation of AC by A2BR was regulated by GRK5, but not GRK2. ß2AR desensitization was attenuated following combined GRK2/GRK5 knockdown, but not by depletion of individual GRKs, arrestins, or by inhibiting PKA. Arrestin3 (but not arrestin2) depletion enhanced A2BR-AC signalling and attenuated A2BR desensitization, while ß2AR-AC signalling was regulated by both arrestin isoforms. This study provides a first demonstration of how different complements of GRK and arrestin proteins contribute to the regulation of signalling and desensitization of these important receptors mediating vasodilator responses in arterial smooth muscle.

GRK2 mediates TCR-induced transactivation of CXCR4 and TCR-CXCR4 complex formation that drives PI3Kγ/PREX1 signaling and T cell cytokine secretion.

The immune system includes abundant examples of biologically-relevant cross-regulation of signaling pathways by the T cell antigen receptor (TCR) and the G protein-coupled chemokine receptor, CXCR4. TCR ligation induces transactivation of CXCR4 and TCR-CXCR4 complex formation, permitting the TCR to signal via CXCR4 to activate a phosphatidylinositol 3,4,5-trisphosphate-dependent Rac exchanger 1 protein (PREX1)-dependent signaling pathway that drives robust cytokine secretion by T cells. To understand this receptor heterodimer and its regulation, we characterized the molecular mechanisms required for TCR-mediated TCR-CXCR4 complex formation. We found that the cytoplasmic C-terminal domain of CXCR4 and specifically phosphorylation of Ser-339 within this region were required for TCR-CXCR4 complex formation. Interestingly, siRNA-mediated depletion of G protein-coupled receptor kinase-2 (GRK2) or inhibition by the GRK2-specific inhibitor, paroxetine, inhibited TCR-induced phosphorylation of CXCR4-Ser-339 and TCR-CXCR4 complex formation. Either GRK2 siRNA or paroxetine treatment of human T cells significantly reduced T cell cytokine production. Upstream, TCR-activated tyrosine kinases caused inducible tyrosine phosphorylation of GRK2 and were required for the GRK2-dependent events of CXCR4-Ser-339 phosphorylation and TCR-CXCR4 complex formation. Downstream of TCR-CXCR4 complex formation, we found that GRK2 and phosphatidylinositol 3-kinase γ (PI3Kγ) were required for TCR-stimulated membrane recruitment of PREX1 and for stabilization of cytokine mRNAs and robust cytokine secretion. Together, our results identify a novel role for GRK2 as a target of TCR signaling that is responsible for TCR-induced transactivation of CXCR4 and TCR-CXCR4 complex formation that signals via PI3Kγ/PREX1 to mediate cytokine production. Therapeutic regulation of GRK2 or PI3Kγ may therefore be useful for limiting cytokines produced by T cell malignancies or autoimmune diseases.

Small-Molecule G Protein-Coupled Receptor Kinase Inhibitors Attenuate G Protein-Coupled Receptor Kinase 2-Mediated Desensitization of Vasoconstrictor-Induced Arterial Contractions.

Vasoconstrictor-driven G protein-coupled receptor (GPCR)/phospholipase C (PLC) signaling increases intracellular Ca2+ concentration to mediate arterial contraction. To counteract vasoconstrictor-induced contraction, GPCR/PLC signaling can be desensitized by G protein-coupled receptor kinases (GRKs), with GRK2 playing a predominant role in isolated arterial smooth muscle cells. In this study, we use an array of GRK2 inhibitors to assess their effects on the desensitization of UTP and angiotensin II (AngII)-mediated arterial contractions. The effects of GRK2 inhibitors on the desensitization of UTP- or AngII-stimulated mesenteric third-order arterial contractions, and PLC activity in isolated mesenteric smooth muscle cells (MSMC), were determined using wire myography and Ca2+ imaging, respectively. Applying a stimulation protocol to cause receptor desensitization resulted in reductions in UTP- and AngII-stimulated arterial contractions. Preincubation with the GRK2 inhibitor paroxetine almost completely prevented desensitization of UTP- and attenuated desensitization of AngII-stimulated arterial contractions. In contrast, fluoxetine was ineffective. Preincubation with alternative GRK2 inhibitors (Takeda compound 101 or CCG224063) also attenuated the desensitization of UTP-mediated arterial contractile responses. In isolated MSMC, paroxetine, Takeda compound 101, and CCG224063 also attenuated the desensitization of UTP- and AngII-stimulated increases in Ca2+, whereas fluoxetine did not. In human uterine smooth muscle cells, paroxetine reversed GRK2-mediated histamine H1 receptor desensitization, but not GRK6-mediated oxytocin receptor desensitization. Utilizing various small-molecule GRK2 inhibitors, we confirm that GRK2 plays a central role in regulating vasoconstrictor-mediated arterial tone, highlighting a potentially novel strategy for blood pressure regulation through targeting GRK2 function.

Role of G protein-coupled receptor kinase 2 in oxidative and nitrosative stress-related neurohistopathological changes in a mouse model of sepsis-associated encephalopathy.

Sepsis-associated encephalopathy (SAE), characterized as diffuse brain dysfunction and neurological manifestations secondary to sepsis, is a common complication in critically ill patients and can give rise to poor outcome, but understanding the molecular basis of this disorder remains a major challenge. Given the emerging role of G protein-coupled receptor 2 (GRK2), first identified as a G protein-coupled receptor (GPCR) regulator, in the regulation of non-G protein-coupled receptor-related molecules contributing to diverse cellular functions and pathology, including inflammation, we tested the hypothesis that GRK2 may be linked to the neuropathogenesis of SAE. When mouse MG6 microglial cells were challenged with lipopolysaccharide (LPS), GRK2 cytosolic expression was highly up-regulated. The ablation of GRK2 by small interfering RNAs (siRNAs) prevented an increase in intracellular reactive oxygen species generation in LPS-stimulated MG6 cells. Furthermore, the LPS-induced up-regulation of inducible nitric-oxide synthase expression and increase in nitric oxide production were negated by GRK2 inhibitor or siRNAs. However, GRK2 inhibition was without effect on overproduction of tumor necrosis factor-α, interleukin (IL)-6, and IL-1ß in LPS-stimulated MG cells. In mice with cecal ligation and puncture-induced sepsis, treatment with GRK2 inhibitor reduced high levels of oxidative and nitrosative stress in the mice brains, where GRK2 expression was up-regulated, alleviated neurohistological damage observed in cerebral cortex sections, and conferred a significant survival advantage to CLP mice. Altogether, these results uncover the novel role for GRK2 in regulating cellular oxidative and nitrosative stress during inflammation and suggest that GRK2 may have a potential as an intriguing therapeutic target to prevent or treat SAE.

ß-Arrestin2 directly or through GRK2 inhibits PKCßII activation in a ubiquitination-dependent manner.

The GRK/ß-arrestin and PKC/PKA mediate the homologous and heterologous regulation of G protein-coupled receptors (GPCRs), respectively. Interaction between the two pathways is one of the most important issues in understanding the regulation of GPCRs. The present study investigated the regulatory effect of GRK2 and ß-arrestins on PKC activation. The roles of GRK2 and ß-arrestins in the functional regulation of PKC were assessed by determining their influence on PKC autophosphorylation and intracellular translocation. Radioligand binding assay was utilized to characterize intracellular trafficking of dopamine D2R, D3R, and ß2 adrenergic receptor (ß2AR). The subdomains involved in the mutual interactions among GRK2, ß-arrestin2, and PKCßII were determined by in vitro binding assay. Various point mutants of key regulatory players were combined with knockdown cells of GRK2, ß-arrestins, and Mdm2 to functionally correlate the biochemical changes with functional outcomes. GRK2 and ß-arrestin2 mutually inhibited the PKCßII autophosphorylation, a hallmark of PKCßII activation. ß-Arrestin2 ubiquitination was required for the inhibitory activities of GRK2 as well as ß-arrestin2. Furthermore, GRK2 facilitated ß-arrestin2 ubiquitination, thus to enhance the inhibitory actions of ß-arrestin2 on PKCßII activity. Aforementioned processes were also involved in the GRK2/ß-arrestin2-mediated inhibition of the D2R, D3R, and ß2AR endocytosis. The present study provides new insights into the intricate interactions between the homologous and heterologous GPCR regulation pathways. In addition, a novel regulatory role of GRK2 was proposed for the ubiquitination of ß-arrestin in the context of the PKC-mediated heterologous regulation of GPCRs.

Involvement of miR-125a in resistance to daunorubicin by inhibiting apoptosis in leukemia cell lines.

In this study, we investigated whether miR-125a participated in the resistance of the leukemia cell lines to the chemotherapeutic agent daunorubicin. Higher expression of miR-125a is correlated with lower treatment response and shorter overall survival in acute leukemia patients. Overexpression of miR-125a induced drug resistance in HL-60, K562, and THP-1cell lines through reducing apoptosis. We also showed that miR-125a mediated daunorubicin resistance in leukemia cell lines through the decrease of GRK2 and Puma which were proved to be direct targets of miR-125a. This study may provide novel therapeutic targets for therapy and improve predictions of therapeutic responses in leukemia to daunorubicin.

Noncanonical Roles of G Protein-coupled Receptor Kinases in Cardiovascular Signaling.

G protein-coupled receptor kinases (GRKs) are classically known for their role in regulating the activity of the largest known class of membrane receptors, which influence diverse biological processes in every cell type in the human body. As researchers have tried to uncover how this family of kinases, containing only 7 members, achieves selective and coordinated control of receptors, they have uncovered a growing number of noncanonical activities for these kinases. These activities include phosphorylation of nonreceptor targets and kinase-independent molecular interactions. In particular, GRK2, GRK3, and GRK5 are the predominant members expressed in the heart. Their canonical and noncanonical actions within cardiac and other tissues have significant implications for cardiovascular function in healthy animals and for the development and progression of disease. This review summarizes what is currently known regarding the activity of these kinases, and particularly the role of GRK2 and GRK5 in the molecular alterations that occur during heart failure. This review further highlights areas of GRK regulation that remain poorly understood and how they may represent novel targets for therapeutic development.

GRK2 Mediates Arginine Vasopressin-Induced Interleukin-6 Production via Nuclear Factor-κB Signaling Neonatal Rat Cardiac Fibroblast.

Interleukin 6 (IL-6), which is elevated in patients with congestive heart failure and acts as both a chronic marker of inflammation and an acute-phase reactant, is associated with myocardial damage. Circulating levels of arginine vasopressin (AVP) are elevated during cardiac stress and could be a factor for cardiac inflammation and fibrosis. Our previous study has shown that AVP promotes the proliferation of neonatal rat cardiac fibroblasts (NRCFs) throughV1A vasopressin receptor-mediated G protein-coupled receptor kinase 2 (GRK2) signaling. In the present study, we investigated the impact of the GRK2-dependent signaling. Using quantitative polymerase chain reaction and enzyme-linked immunosorbent assay, we measured the levels of interleukin-6 (IL-6) mRNA and protein in NRCFs, respectively. Manipulation of GRK2 activation either pharmacologically or through overexpression of GRK2-ct was used to determine the role of GRK2 in regulating the effects of AVP on IL-6 production. Phosphorylation and activation of nuclear factor κ-B (NF-κB) evoked by AVP stimulation were measured by immunoblot and NF-kB luciferase reporter gene transfected in NRCFs, respectively. Present studies have found that: 1) AVP increased the level of IL-6 protein and mRNA in a dose- and time-dependent manner in NRCFs; 2) inhibition of GRK2 abolished the AVP-induced IL-6 production and NF-κB activation; and 3) blocking NF-κB signaling using the pharmacologic approach diminished AVP-induced IL-6 production. In summary, AVP induces IL-6 production of NRCFs by activating V1A receptor signaling via a GRK2/NF-κB pathway. These findings provide a possible molecular mechanism for inflammation that occurs in heart failure and other types of cardiac stress.

Cardiac Fibroblast GRK2 Deletion Enhances Contractility and Remodeling Following Ischemia/Reperfusion Injury.

RATIONALE: G protein-coupled receptor kinase 2 (GRK2) is an important molecule upregulated after myocardial injury and during heart failure. Myocyte-specific GRK2 loss before and after myocardial ischemic injury improves cardiac function and remodeling. The cardiac fibroblast plays an important role in the repair and remodeling events after cardiac ischemia; the importance of GRK2 in these events has not been investigated. OBJECTIVE: The aim of this study is to elucidate the in vivo implications of deleting GRK2 in the cardiac fibroblast after ischemia/reperfusion injury. METHODS AND RESULTS: We demonstrate, using Tamoxifen inducible, fibroblast-specific GRK2 knockout mice, that GRK2 loss confers a protective advantage over control mice after myocardial ischemia/reperfusion injury. Fibroblast GRK2 knockout mice presented with decreased infarct size and preserved cardiac function 24 hours post ischemia/reperfusion as demonstrated by increased ejection fraction (59.1±1.8% versus 48.7±1.2% in controls; P<0.01). GRK2 fibroblast knockout mice also had decreased fibrosis and fibrotic gene expression. Importantly, these protective effects correlated with decreased infiltration of neutrophils to the ischemia site and decreased levels of tumor necrosis factor-α expression and secretion in GRK2 fibroblast knockout mice. CONCLUSIONS: These novel data showing the benefits of inhibiting GRK2 in the cardiac fibroblast adds to previously published data showing the advantage of GRK2 ablation and reinforces the therapeutic potential of GRK2 inhibition in the heart after myocardial ischemia.

RalA employs GRK2 and ß-arrestins for the filamin A-mediated regulation of trafficking and signaling of dopamine D2 and D3 receptor.

Filamin A (FLNA) is known to act as platform for the signaling and intracellular trafficking of various GPCRs including dopamine D2 and D3 receptors (D2R, D3R). To understand molecular mechanisms involved in the FLNA-mediated regulation of D2R and D3R, comparative studies were conducted on the signaling and intracellular trafficking of the D2R and D3R in FLNA-knockdown cells, with a specific focus on the roles of the proteins that interact with FLNA and the D2R and D3R. Lowering the level of cellular FLNA caused an elevation in RalA activity and resulted in selective interference with the normal intracellular trafficking and signaling of the D2R and D3R, through GRK2 and ß-arrestins, respectively. Knockdown of FLNA or coexpression of active RalA interfered with the recycling of the internalized D2R and resulted in the development of receptor tolerance. Active RalA was found to interact with GRK2 to sequester it from D2R. Knockdown of FLNA or coexpression of active RalA prevented D3R from coupling with G protein. The selective involvement of GRK2- and ß-arrestins in the RalA-mediated cellular processes of the D2R and D3R was achieved via their different modes of interactions with the receptor and their distinct functional roles in receptor regulation. Our results show that FLNA is a multi-functional protein that acts as a platform on which D2R and D3R can interact with various proteins, through which selective regulation of these receptors occurs in combination with GRK2 and ß-arrestins.

Critical role for Epac1 in inflammatory pain controlled by GRK2-mediated phosphorylation of Epac1.

cAMP signaling plays a key role in regulating pain sensitivity. Here, we uncover a previously unidentified molecular mechanism in which direct phosphorylation of the exchange protein directly activated by cAMP 1 (EPAC1) by G protein kinase 2 (GRK2) suppresses Epac1-to-Rap1 signaling, thereby inhibiting persistent inflammatory pain. Epac1(-/-) mice are protected against inflammatory hyperalgesia in the complete Freund's adjuvant (CFA) model. Moreover, the Epac-specific inhibitor ESI-09 inhibits established CFA-induced mechanical hyperalgesia without affecting normal mechanical sensitivity. At the mechanistic level, CFA increased activity of the Epac target Rap1 in dorsal root ganglia of WT, but not of Epac1(-/-), mice. Using sensory neuron-specific overexpression of GRK2 or its kinase-dead mutant in vivo, we demonstrate that GRK2 inhibits CFA-induced hyperalgesia in a kinase activity-dependent manner. In vitro, GRK2 inhibits Epac1-to-Rap1 signaling by phosphorylation of Epac1 at Ser-108 in the Disheveled/Egl-10/pleckstrin domain. This phosphorylation event inhibits agonist-induced translocation of Epac1 to the plasma membrane, thereby reducing Rap1 activation. Finally, we show that GRK2 inhibits Epac1-mediated sensitization of the mechanosensor Piezo2 and that Piezo2 contributes to inflammatory mechanical hyperalgesia. Collectively, these findings identify a key role of Epac1 in chronic inflammatory pain and a molecular mechanism for controlling Epac1 activity and chronic pain through phosphorylation of Epac1 at Ser-108. Importantly, using the Epac inhibitor ESI-09, we validate Epac1 as a potential therapeutic target for chronic pain.

G-protein-coupled receptor kinase 2 and endothelial dysfunction: molecular insights and pathophysiological mechanisms.

Smooth muscle cells (SMC) and endothelial cells are the major cell types in blood vessels. The principal function of vascular SMC in the body is to regulate blood flow and pressure through contraction and relaxation. The endothelium performs a crucial role in maintaining vascular integrity by achieving whole-organ metabolic homeostasis via the production of factors associated with vasoconstriction or vasorelaxation. In this review, we have focused on the production of nitric oxide (NO), a vasorelaxation factor. The extent of NO production represents a key marker in vascular health. A decrease in NO is capable of inducing pathological conditions associated with endothelial dysfunction, such as obesity, diabetes, cardiovascular disease, and atherosclerosis. Recent studies have strongly implicated the involvement of G-protein-coupled receptor kinase 2 (GRK2) in the progression of cardiovascular disease. Vasculature which is affected by insulin resistance and type 2 diabetes expresses high levels of GRK2, which may induce endothelial dysfunction by reducing intracellular NO. GRK2 activation also induces changes in the subcellular localization of GRK2 itself and also of ß-arrestin 2, a downstream protein. In this review, we describe the pathophysiological mechanisms of insulin resistance and diabetes, focusing on the signal transduction for NO production via GRK2 and ß-arrestin 2, providing novel insights into the potential field of translational investigation in the treatment of diabetic complications.

[The Role of GRK2 and Its Potential as a New Therapeutic Target in Diabetic Vascular Complications].

A decrease in nitric oxide (NO) production may induce pathological conditions associated with endothelial dysfunction and diabetes. Although a decrease in NO production caused by impaired Akt/endothelial nitric oxide synthesis (eNOS) signaling has been demonstrated at the aorta in the presence of diabetic vascular complications, little is known regarding the details of the mechanism. We identified G-protein-coupled receptor kinase 2 (GRK2) as a critical factor in diabetic endothelial dysfunction. GRK2 plays a role in many physiological functions including regulation of G-protein-coupled receptors (GPCRs). We found that the vasculature affected by type 2 diabetes expresses high levels of GRK2, which may induce endothelial dysfunction caused by impaired Akt/eNOS signaling. GRK2 activation also induces changes in the subcellular localization of GRK2 and ß-arrestin 2, a downstream protein, from the cytosol to membrane. In mouse aorta GRK2 may be, on translocation, a key negative regulator and an important regulator of ß-arrestin 2/Akt/eNOS signaling, which has been implicated in diabetic endothelial dysfunction. Furthermore, in the aortic membrane of type 2 diabetic model mice under insulin stimulation, the impaired Akt/eNOS signaling was improved by a selective GRK2 inhibitor. These results suggest that in diabetes the GRK2 inhibitor ameliorates vascular endothelial dysfunction via Akt/eNOS signaling by inhibiting GRK2 activity and enhancing ß-arrestin 2 translocation to the membrane under GPCR or non-GPCR stimulation, thereby contributing to blood pressure- and blood glucose-lowering effects. We propose that the GRK2 inhibitor may be a promising therapeutic target for cardiovascular complications in type 2 diabetes.