Role Of GRK2 Research Articles

Selective deletion of GRK2 alters psychostimulant-induced behaviors and dopamine neurotransmission.

GRK2 is a G protein-coupled receptor kinase (GRK) that is broadly expressed and is known to regulate diverse types of receptors. GRK2 null animals exhibit embryonic lethality due to a severe developmental heart defect, which has precluded the study of this kinase in the adult brain. To elucidate the specific role of GRK2 in the brain dopamine (DA) system, we used a conditional gene knockout approach to selectively delete GRK2 in DA D1 receptor (D1R)-, DA D2 receptor (D2R)-, adenosine 2A receptor (A2AR)-, or DA transporter (DAT)-expressing neurons. Here we show that select GRK2-deficient mice display hyperactivity, hyposensitivity, or hypersensitivity to the psychomotor effects of cocaine, altered striatal signaling, and DA release and uptake. Mice with GRK2 deficiency in D2R-expressing neurons also exhibited increased D2 autoreceptor activity. These findings reveal a cell-type-specific role for GRK2 in the regulation of normal motor behavior, sensitivity to psychostimulants, dopamine neurotransmission, and D2 autoreceptor function.

Abstract 074: GRK5 Increases Cardiac NFAT Transcriptional Activity

Introduction: G protein-coupled receptor (GPCR) kinase-2 and -5 (GRK2 and GRK5) have been shown to be up-regulated in failing human myocardium. While the canonical role of GRKs is to desensitize receptors via phosphorylation, it has been shown that GRK5, unlike GRK2, can reside in the nucleus of cardiomyocytes and exert GPCR-independent effects that promote maladaptive cardiac hypertrophy and heart failure (HF). Previously, our lab has shown that GRK5 increases hypertrophic gene transcription through the phosphorylation of histone deacetylase 5 (HDAC5) and subsequent disinhibition of the transcription factor, myocyte enhancer factor-2 (MEF2). Use of GRK5 knockout (KO) mice has recently proved that GRK5 is indeed a physiological HDAC kinase during hypertrophic stress. Through experiments described below we have now identified the nuclear factor of activated T cells (NFAT) pathway as another potential target of GRK5 during cardiac hypertrophy that may play a role in its facilitation of HF. Methods and Results: Cardiac-specific NFAT-luciferase reporter mice were crossed with mice that overexpress wild-type GRK5 in myocytes to determine the role for GRK5 in NFAT signaling. These double-transgenic mice along with controls were stressed using the hypertrophic α 1 -adrenergic agonist phenylephrine (PE) as well as ventricular pressure-overload via transverse aortic constriction (TAC). NFAT activity was determined by both in vivo and ex vivo luciferase assays as well as RT-PCR for the NFAT target gene RCAN. Cardiac specific GRK5 overexpression leads to an increase in NFAT activity in the basal state as well as after both forms of hypertrophic stress. This was reproduced in cultured myocytes using a NFAT-reporter construct. Overexpression of a mutant GRK5 that cannot enter the nucleus appears to not activate NFAT demonstrating a nuclear-dependence. Consistent with this, GRK5 KO mice after TAC showed a decrease in the up-regulation of RCAN transcription as compared to wild-type mice. Studies are ongoing to determine the mechanism of GRK5’s regulation of cardiac NFAT activity. Conclusions: This study provides another non-canonical role for GRK5 in activating the hypertrophic NFAT pathway in cardiomyocytes.

Abstract 075: Novel Role for GRK2 in Cardiac Hypertrophy and Heart Failure

During heart failure (HF), cardiac levels and activity of the G protein-coupled receptor (GPCR) kinase (GRK) GRK2 are elevated, increasing phosphorylation, desensitization and down-regulation of β-adrenergic receptors (βARs) and other cardiac GPCRs. Increased GRK2 has been shown to participate in adverse remodeling and contractile dysfunction during HF, while GRK2 inhibition via a carboxy-terminal peptide, βARKct, enhances heart function and can prevent HF development. Mounting evidence supports the idea of a dynamic GRK2 “interactome” in which GRK2 can uncouple GPCRs via novel protein-protein interactions. Several novel GRK2 interacting partners are important for adaptive and maladaptive myocyte growth including Gq, the signaling trigger for maladaptive cardiac hypertrophy, leading to HF. Importantly, GRK2 contains a putative amino-terminal Regulator of G protein Signaling (RGS) domain (termed βARK-RGS). This domain directly interacts with Gq and appears to inhibit signaling without altering Gq enzymatic activity. Therefore, this domain of GRK2 may alter hypertrophic responses in the heart and represent a novel role for GRK2 and also a potential therapeutic target to limit maladaptive cardiac hypertrophy. We have begun to address this by generation of novel transgenic mice with cardiac-specific expression of the RGS domain of GRK2. Data from mice with cardiac expression of βARK-RGS demonstrate anti-hypertrophic effects in a trans-aortic constriction (TAC) model of pressure overload hypertrophy. Echocardiographic analysis post-TAC revealed reduced left ventricular posterior wall thickness in βARK-RGS compared to non-transgenic littermate controls (1.34 vs 1.57 mm LVPWd at 4 weeks). RT-PCR analysis found decreased hypertrophic factor transcripts, such as ANF for which the nearly 18-fold increase post TAC was completely inhibited in βARK-RGS mice. Other hypertrophic phenotypic markers have been studied and mechanistic characterization is underway. These data support our hypothesis that the RGS domain of GRK2 may serve as a non-canonical inhibitor of Gq-mediated hypertrophic signaling in the heart and highlight how this research may pave the way for novel GRK2-based therapeutic approaches to prevent hypertrophy and HF.

346 Role of GRK2 in Developing Vasculature and Pathological Angiogenesis

346 Role of GRK2 in Developing Vasculature and Pathological Angiogenesis

Roles of GRK2 in Cell Signaling Beyond GPCR Desensitization: GRK2-HDAC6 Interaction Modulates Cell Spreading and MotilityA Presentation from the Cell Signaling Networks Conference and 13th IUBMB Conference, Mérida, Yucatán, México, 22 to 27 October 2011.

G protein-coupled receptor kinase 2 (GRK2) is a ubiquitous, essential protein kinase that is emerging as an integrative node in many signaling networks. Moreover, changes in GRK2 abundance and activity have been identified in several inflammatory, cardiovascular disease, and tumor contexts, suggesting that those alterations may contribute to the initiation or development of pathologies. GRKs were initially identified as key players in the desensitization and internalization of multiple G protein-coupled receptors (GPCRs), but GRK2 also phosphorylates several non-GPCR substrates and dynamically associates with a variety of proteins related to signal transduction. Ongoing research in our laboratory is aimed at understanding how specific GRK2 interactomes are orchestrated in a stimulus-, context-, or cell type-specific manner. We have recently identified an interaction between GRK2 and histone deacetylase 6 (HDAC6) that modulates cell spreading and motility. HDAC6 is a major cytoplasmic a-tubulin deacetylase that is involved in cell motility and adhesion. GRK2 dynamically and directly associates with and phosphorylates HDAC6 to stimulate its a-tubulin deacetylase activity at specific cellular localizations, such as the leading edge of migrating cells, thus promoting local tubulin deacetylation and enhanced motility. GRK2-HDAC6-mediated regulation of tubulin acetylation also modulates cellular spreading. This GRK2-HDAC6 functional interaction may have important implications in pathological contexts related to epithelial cell migration.

Abstract 1234: GRK2 negatively regulates IGF-1R signaling pathway and cyclins in HepG2 cells

Abstract G protein-coupled receptor kinase 2 (GRK2) plays a central role in the regulation of a variety of signaling pathways associated with many important cellular processes. Alternation of GRK2 expression has profound effects on cell physiological functions and induces diseases such as heart failure, rheumatoid arthritis, and cancer. In this study, we investigated the role of GRK2 in human hepatocellular carcinoma (HCC) HepG2 cells. We created a GRK2 knockdown cell line using a lentivirus containing GRK2 specific shRNA. Under IGF-1 (50 ng/ml) stimulation, HepG2 cells with reduced GRK2 expression showed increased tyrosine phosphorylation of IRS1 at the residue 612 and increased phosphorylation of Akt. We then treated the cells with gefitinib, an EGFR inhibitor. We found HepG2 cells displayed gefitinib sensitiveness with the increase of drug concentration. Moreover, gefitinib treated cells with reduced expression of GRK2 showed decreased phosphorylation of Akt in a dose dependent manner, but had relatively more IGF-1R expression. However, HepG2 cells with reduced expression of GRK2 did not display any growth difference in culture as compared with the scramble control cells. We further detected that reduced expression of GRK2 induced a small G2/M phase arrest by overexpressing cyclin A, B1, and E. DNA microarray analysis showed decreased transcription levels of IGF binding protein 1 and 4, which were confirmed by quantitative PCR analysis. Our results demonstrated that GRK2 has a role in the regulation of IGF-1R signaling pathway and involves in cell cycle progression in HCC HepG2 cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1234. doi:1538-7445.AM2012-1234

Abstract P129: G Protein--Coupled Receptor Kinase-2 (GRK2) Is a Novel Regulator of Collagen Synthesis in Adult Human Cardiac Fibroblasts

Cardiac fibroblasts (CF) make up 70% of the total cell number in the heart and play a critical role in regulating normal myocardial function and in adverse remodeling following myocardial infarction. Recent studies have shown that increased intracellular cAMP can inhibit CF transformation and collagen synthesis in adult rat CF; however, mechanisms by which cAMP production is regulated in CF have not been elucidated. The objective of this study was to investigate the potential role of GRK2 in modulating CF transformation to myofibroblasts and collagen synthesis in adult human CF isolated from normal and failing left ventricles. CF isolated from failing ventricles showed a significant increase in expression of collagen I, III and VI compared with controls. α-SMA was increased 2-fold over controls, consistent with CF transformation to myofibroblasts. Baseline collagen synthesis was elevated 2-fold in failing CF and was not inhibited by isoproterenol (ISO)-stimulation in contrast to normal controls. β-adrenergic receptor (β-AR) signaling was markedly uncoupled in failing CF as assessed by basal and ISO-stimulated cAMP production. The primary mechanism appears to be a 2.5-fold increase in GRK2 activity as GRK2 phosphorylates and uncouples agonist-occupied β-ARs. Overexpression of GRK2 in normal CF recapitulated a heart failure phenotype with minimal inhibition of collagen synthesis following ISO stimulation. In contrast, siRNA-mediated knockdown of GRK2 expression in normal CF enhanced cAMP production and led to greater β-agonist-mediated inhibition of basal and TGFβ-stimulated collagen synthesis versus control. Inhibition of GRK2 activity by adenoviral-mediated βARKct expression or GRK2 knockdown in failing CF led to a significant decline in collagen and α-SMA expression. GRK2 inhibition restored β-AR signaling and ISO-stimulated inhibition of collagen synthesis and also significantly decreased collagen synthesis in response to TGFβ stimulation. In conclusion, GRK2 appears to play a significant role in regulating CF transformation and collagen synthesis in adult human CF and increased activity of this kinase may be an important mechanism of maladaptive ventricular remodeling as mediated by cardiac fibroblasts.

Mitochondrial localization unveils a novel role for GRK2 in organelle biogenesis

Metabolic stimuli such as insulin and insulin like growth factor cause cellular accumulation of G protein coupled receptor kinase 2 (GRK2), which in turn is able to induce insulin resistance. Here we show that in fibroblasts, GRK2 is able to increase ATP cellular content by enhancing mitochondrial biogenesis; also, it antagonizes ATP loss after hypoxia/reperfusion. Interestingly, GRK2 is able to localize in the mitochondrial outer membrane, possibly through one region within the RGS homology domain and one region within the catalytic domain. In vivo, GRK2 removal from the skeletal muscle results in reduced ATP production and impaired tolerance to ischemia. Our data show a novel sub-cellular localization of GRK2 in the mitochondria and an unexpected role in regulating mitochondrial biogenesis and ATP generation.

Abstract 603: Overexpression of G protein-coupled receptor kinase 2 in hepatocellular carcinoma cells suppresses proliferation by inducing G2/M phase arrest

Abstract G protein-coupled receptor kinase 2 (GRK2) was originally identified as a key player in G protein-coupled receptors desensitization. A growing body of evidence has demonstrated that GRK2 may have broader ‘effector” functions by interacting with a variety of non-receptor signaling proteins and involves in the regulation of many cellular processes such as proliferation, cell cycle progression and migration. In this study, we investigated the role of GRK2 in human hepatocellular carcinoma (HCC) cells. We screened five HCC cell lines and found that Huh7 and Mahlavu had the highest and lowest of basal GRK2 expression levels respectively. We then overexpressed wild type (WT) GRK2 and its kinase-dead mutant (K220R) in these two HCC cells using recombinant adenoviruses. We found that only WT GRK2 overexpression but not the K220R mutant overexpression significantly reduced cell viability. We thus performed BrdU incorporation assay and found that only WT GRK2 overexpression reduced cell proliferation. We further examined this anti-proliferative function by propidium iodide staining to analyze cell cycle progression. Our data showed that enhanced WT GRK2 induced cell cycle arrest at G2/M phase, from 21.4% to 41.8% in Huh7 and 25.9% to 48.4% in Mahlavu, respectively. Furthermore, our western blotting results showed that overexpression of GRK2 induced the up-regulation of p53 (Ser15) phosphorylation and Cyclin B1 in a dose dependent manner. Our results indicated that overexpression of GRK2 had a profound suppressive effects on HCC cell growth and suggested that gene transfer of GRK2 may open a new therapeutic option for the treatment of human HCC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 603. doi:10.1158/1538-7445.AM2011-603

Highlights From The Literature

Highlights From The Literature

GRK5 regulates NFkB transcription activity in endothelial cells

Recent studies suggest that G protein-coupled receptor kinases (GRKs) play multiple roles in regulating signalling. In particular, GRK5 is able to enter the nucleus through nuclear localization (NLS) and exporting (NES) sequences. Our hypothesis is that GRK5 regulates transcription events in the nucleus, in particular NFkB activity. In bovine aortic endothelial cells GRK5 associates to IkB, a NFkB inhibitor and such interaction increases in presence of isoproterenol, β adrenergic agonist, 10 M. GRK5 overexpression causes nuclear accumulation of IkB, suggesting a role for GRK5 to inhibit NFkB transcriptional activity. To confirm the functional implication of such interaction, we used luciferase as reporter gene under the control of a NFkB-dependent promoter. GRK5 overexpression inhibits NFkB activity both in presence and absence of isoproterenol, TNFα and LPS. These data suggest an universal role of GRK5 to inhibit NFkB activity. To evaluate the role of NLS and NES in GRK5IkB interaction, we overexpressed GRK5 mutants lacking either NLS or NES sequences (GRK5ΔNLS and GRK5ΔNES). Both GRK5ΔNLS and GRK5ΔNES overexpression results in inhibition of NFkB activity (GRK5: −73±16%; GRK5ΔNLS: 42±6%; GRK5ΔNES: −67±12%; p<0.01 vs. control) and nuclear accumulation. Immunoprecipitation studies with overexpression of small peptides reproducing single domains of GRK5 (N-CAM, RGS, Catalytic domain, C-CAM) suggest that RGS is the GRK5 domain that associates to IkB. In conclusion, these results reveal a novel role of GRK5 as modulator of NFkB transcriptional activity that will be object of future investigations in cardiovascular disease models.

Scoring of predicted GRK2 phosphorylation sites in Nedd4-2

Epithelial Na(+) channels (ENaC) mediate the transport of sodium (Na) across epithelia in the kidney, gut and lungs and are required for blood pressure regulation. They are inhibited by ubiquitin protein ligases, such as Nedd4-2. These ligases bind to proline-rich motifs (PY motifs) present in the C-termini of ENaC subunits. Loss of this inhibition leads to hypertension. We have previously reported that ENaC channels are maintained in the active state by the G protein coupled receptor kinase, GRK2. The enzyme has been implicated in the development of essential hypertension [R. D. Feldman (2002) Mol. Pharmacol., 61, 707-709]. Additional findings in our lab pointed towards a possible role for GRK2 in the phosphorylation and inactivation of Nedd4-2. We have predicted GRK2 phosphorylation sites on Nedd4-2 by combining sequence analysis, homology modeling and surface accessibility calculations. A total of 24 potential phosphorylation sites were predicted by sequence analysis. Of these, 16 could be modeled using homology modeling and 6 of these were found to have sufficient surface exposure to be accessible to the GRK2 enzyme responsible for the phosphorylation of Nedd4-2. The method provides an ordered list of the most probable GRK2 phosphorylation sites on Nedd4-2 providing invaluable guidance to future experimental studies aimed at mutating certain Nedd4-2 residues in order to prevent phosphorylation by GRK2. The method developed could be applied in a wide variety of biological applications involving the binding of one molecule to a protein. The relative effectiveness of the technique is determined mainly by the quality of the homology model built for the protein of interest. jarthur@med.usyd.edu.au

Different G protein-coupled receptor kinases govern G protein and β-arrestin-mediated signaling of V2 vasopressin receptor

Signaling through beta-arrestins is a recently appreciated mechanism used by seven-transmembrane receptors. Because G protein-coupled receptor kinase (GRK) phosphorylation of such receptors is generally a prerequisite for beta-arrestin binding, we studied the roles of different GRKs in promoting beta-arrestin-mediated extracellular signal-regulated kinase (ERK) activation by a typical seven-transmembrane receptor, the Gs-coupled V2 vasopressin receptor. Gs- and beta-arrestin-mediated pathways to ERK activation could be distinguished with H89, an inhibitor of protein kinase A, and beta-arrestin 2 small interfering RNA, respectively. The roles of GRK2, -3, -5, and -6 were assessed by suppressing their expression with specific small interfering RNA sequences. By using this approach, we demonstrated that GRK2 and -3 are responsible for most of the agonist-dependent receptor phosphorylation, desensitization, and recruitment of beta-arrestins. In contrast, GRK5 and -6 mediated much less receptor phosphorylation and beta-arrestin recruitment, but yet appeared exclusively to support beta-arrestin 2-mediated ERK activation. GRK2 suppression actually increased beta-arrestin-stimulated ERK activation. These results suggest that beta-arrestin recruited in response to receptor phosphorylation by different GRKs has distinct functional potentials.