GRK2 Knockdown Research Articles

The short third intracellular loop and cytoplasmic tail of bitter taste receptors provide functionally relevant GRK phosphorylation sites in TAS2R14

For most G protein–coupled receptors, the third intracellular loop (IL3) and carboxy-terminal tail (CT) are sites for G protein–coupled receptor kinase (GRK)–mediated phosphorylation, leading to β-arrestin binding and agonist-specific desensitization. These regions of bitter taste receptors (TAS2Rs) are extremely short compared with the superfamily, and their function in desensitization is unknown. TAS2R14 expressed on human airway smooth muscle cells relax the cell, suggesting a novel target for bronchodilators. To assess IL3 and CT in agonist-promoted TAS2R14 desensitization (tachyphylaxis), we generated fusion proteins of both the WT sequence and Ala substituted for Ser/Thr in the IL3 and CT sequences. In vitro, activated GRK2 phosphorylated WT IL3 and WT CT proteins but not Ala-substituted forms. TAS2R14s with mutations in IL3 (IL-5A), CT (CT-5A), and in both regions (IL/CT-10A) were expressed in human embryonic kidney 293T cells. IL/CT-10A and CT-5A failed to undergo desensitization of the intracellular calcium response compared with WT, indicating that functional desensitization by GRK phosphorylation is at residues in the CT. Desensitization of TAS2R14 was blocked by GRK2 knockdown in human airway smooth muscle cells. Receptor:β-arrestin binding was absent in IL/CT-10A and CT-5A and reduced in IL-5A, indicating a role for IL3 phosphorylation in the β-arrestin interaction for this function. Agonist-promoted internalization of IL-5A and CT-5A receptors was impaired, and they failed to colocalize with early endosomes. Thus, agonist-promoted functional desensitization of TAS2R14 occurs by GRK phosphorylation of CT residues and β-arrestin binding. However, β-arrestin function in the internalization and trafficking of the receptor also requires GRK phosphorylation of IL3 residues.

GRK2 Suppresses Hepatocellular Carcinoma Metastasis and Invasion Through Down-Regulation of Prostaglandin E Receptor 2.

BackgroundHepatocellular carcinoma (HCC) is an aggressive form of human liver cancer and the fifth most common malignancy worldwide. Novel effective treatment strategies for HCC are urgently in clinical because of its poor response to conventional therapies. G protein-coupled receptor kinases (GRKs), including GRK2 and GRK3, are known that involves in various essential cellular processes and regulates numerous signaling pathways. However, the role of GRK2/3 in invasion and metastasis of HCC still remains unclear.Materials and MethodsImmunohistochemistry, Western blot, laser confocal microscopy and qRT-PCR were used to detect the expression of GRK2/3 and EP2 in liver tissues of HCC patients and DEN-induced HCC mice. Wound healing and transwell assay were applied to measure the migration and invasion of HCC cells after transfected with GRK2 siRNA. The downstream pathway of Akt and ERK was verified by Western blot.ResultsThe expression of GRK2 was significantly decreased, while GRK3 was not significantly changed in HCC tissues compared with noncancerous tissues of HCC patients. Moreover, GRK2 expression was reduced during liver tumorigenesis in diethylnitrosamine-induced liver tumor model. In addition, our in vitro study showed that GRK2 expression was gradually decreased with increasing HCC cell line metastatic potential, and GRK2 knockdown significantly promoted the migration and invasion of HCC cells. Furthermore, low GRK2 expression was associated with increased expression of EP2 receptor translocation to HCC cell membrane, and the activation of Akt pathway.ConclusionThese data suggest that GRK2 inhibits HCC metastasis and invasion may be through regulating EP2 receptor translocation, and this effect appears to be mediated by Akt pathway.

GRK2 promotes growth of medulloblastoma cells and protects them from chemotherapy-induced apoptosis

G-protein coupled receptor kinase 2 (GRK2; ADRBK1, BARK1) is most known as a regulator of G-protein coupled receptors. However, GRK2 also has other functions. Medulloblastomas are the most common malignant brain cancers in children. GRK2 has not been implicated in medulloblastoma biology. Here we report that GRK2 knockdown slowed cell growth, diminished proliferation, and enhanced cisplatin- and etoposide-induced apoptosis in medulloblastoma cell lines UW228-2 and Daoy. Reciprocally, GRK2 overexpression attenuated apoptosis induced by these chemotherapy drugs. Cisplatin and etoposide increased phosphorylation of AKT (S473) and GRK2 knockdown mitigated this increase. Cisplatin and etoposide attenuated ERK phosphorylation, but GRK2 knockdown did not alter this effect. Wildtype GRK2 reversed the increase in cisplatin- and etoposide-induced apoptosis caused by GRK2 knockdown. GRK2-K220R (kinase dead) and GRK2-S670A (unphosphorylated, constitutively active) conferred protection from cisplatin that was similar to wildtype GRK2, suggesting that this protection may be mediated though a kinase-independent activity of GRK2. These data demonstrate that GRK2 contributes to proliferation and survival of these medulloblastoma cell lines and to their protection from cisplatin- and etoposide-induced apoptosis.

Abstract 2053: Targeting GRK2 increases chemotherapy-induced apoptosis in medulloblastoma

Abstract Background: G protein receptor kinase-2 (GRK2; ADRBK1) belongs to the family of G protein-coupled receptor (GPCR) kinases (GRKs). GRK2 phosphorylates GPCR and non-GPCR targets including cell surface receptors, cytoskeletal and mitochondrial proteins and transcription factors. It also functions in embryonic development, heart function, metabolism, and some cancer models. Here we hypothesized that targeting GRK2 will sensitizes cancer cells to therapy. We tested this in medulloblastoma, the most common malignant brain tumor in children. Methods: siRNA transfections, shRNA viral infections, SDS-PAGE, western blotting, flow cytometry. Cells: UW228 and DAOY medulloblastoma. Results: We found that GRK2 inhibition promotes anti-tumor effects of chemotherapeutic drugs cisplatin and etoposide in UW228 and DAOY medulloblastoma cell lines. GRK2 silencing increased apoptosis induced by cisplatin and etoposide and GRK2 overexpression decreased apoptosis induced by these drugs in UW228 and DAOY. Time dependent studies revealed that GRK2 overexpression delayed cisplatin- and etoposide-induced apoptosis measured by the induction of Annexin V positive population and by PARP-1 cleavage. GRK2 mutants, GRK2 K220R (kinase deficient mutant) and GRK2 S670A (cannot be phosphorylated) were similar to wildtype GRK2 in delaying the cisplatin and etoposide induced apoptosis. GRK2 knockdown antagonized serum induced AKT and ERK phosphorylation in both lines whereas GRK2 overexpression promoted phosphorylation, suggesting that GRK2 is important in medulloblastoma survival pathways. Conclusions: These results demonstrate that targeting GRK2 enhances cisplatin- and etoposide-induced apoptosis in medulloblastoma cell lines and suggests that GRK2 inhibitors may enhance responsiveness of medulloblastoma to chemotherapy. Citation Format: Anup S. Pathania, Xiuhai Ren, Min Mahdi, Gregory M. Shackleford, Anat Erdreich-Epstein. Targeting GRK2 increases chemotherapy-induced apoptosis in medulloblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2053.

High-glucose induces cardiac myocytes apoptosis through Foxo1 /GRK2 signaling pathway

High glucose-induced cardiac myocytes apoptosis has been well demonstrated, but the mechanism remains unknown. In this study, we found that exposure of cardiac H9c2 cells to high glucose promoted Foxo1 and GRK2 expression, and induced autophagy. Further investigation showed that high glucose simultaneously increased the expression of cytoplasmic and nuclear Foxo1. Inhibition of Foxo1 reduced GRK2 expression and blocked autophagy, enhancing high glucose-induced apoptosis. GRK2 knockdown did not significantly affect Foxo1 expression and autophagy, but attenuated high glucose-induced apoptosis. Intriguingly, GRK2 knockdown reduced ROS generation. NAC treatment not only reduced the levels of cytoplasmic and nuclear Foxo1, but also inhibited GRK2 expression and autophagy, remarkably reducing high glucose-induced apoptosis. Inhibition of autophagy did not notably affect the expression of Foxo1 and GRK2, but enlarged high glucose-induced apoptosis. ChIP assay and Luciferase reporter assay confirmed that Foxo1 positively regulated GRK2 transcription. These results suggested that Foxo1 was involved in glucose-induced apoptosis by regulating GRK2 expression and autophagy.

CP-25 reverses prostaglandin E4 receptor desensitization-induced fibroblast-like synoviocyte dysfunction via the G protein-coupled receptor kinase 2 in autoimmune arthritis.

Paeoniflorin-6'-O-benzene sulfonate (CP-25) is a novel compound derived from paeoniflorin that has been demonstrated to have therapeutic effects in a rat model of rheumatoid arthritis (RA). However, the underlying mechanism has not been elucidated to date. We explored this mechanism in the present study by treating rats with adjuvant arthritis (AA) with CP-25. We found that the membrane EP4 protein level was downregulated; whereas, GRK2 was upregulated, in fibroblast-like synoviocyte (FLS)s of AA rats. Prostaglandin (PGE)2 stimulated FLS proliferation and enhanced the membrane EP4 receptor protein level; the latter was reversed by the administration of an EP4 receptor agonist, whereas the membrane GRK2 protein level gradually increased. The changes in the EP4 receptor and GRK2 expression were enhanced by TNF-α, and the former was accompanied by an alteration in the cyclic (c)AMP level. The EP4 receptor agonist stimulation increased the association between GRK2 and the EP4 receptor. GRK2 knockdown abrogated the abnormalities in FLS proliferation. The CP-25 treatment (100 mg/kg) suppressed joint inflammation with an efficacy that was similar to that of methotrexate. This finding was associated with EP4 upregulation and GRK2 downregulation in FLSs. Thus, GRK2 plays an important role in the abnormal FLS proliferation observed in AA possibly by promoting EP4 receptor desensitization and decreasing the cAMP level. Our results demonstrate that CP-25 has therapeutic potential for the treatment of human RA via GRK2 regulation.

G protein Coupled Receptor Kinase 2 Levels Regulate Differential Internalization of C-C Chemokine Receptor 7 (CCR7) in Primary Mouse T Cells

Abstract G protein-coupled receptor (GPCR) signaling can be regulated by phosphorylation via GPCR kinases (GRKs) and recruitment of b-arrestins. The C-C chemokine receptor 7 (CCR7) GPCR, has two ligands, CCL19 and CCL21 that promote chemotactic migration of CCR7 expressing immune cells, including naïve T cells. Distinct signaling pathways are activated in T cells depending upon which ligand is bound to the CCR7. It remains unclear to what extent GRKs contribute to differential internalization of ligand-bound CCR7 and subsequent signaling that controls T cell migration. We used naïve primary T cells from wild type C57 BL/6 mice (WT) or syngeneic mice heterozygous for the GRK2 gene that express 50% GRK2 protein levels compared to WT mice. T cells were incubated with 40 nM of CCL19 or CCL21 for up to 8 minutes. Incubation with CCL19, but not CCL21, led to CCR7 internalization; however, both ligands facilitated rapid, sustained CCR7 internalization in GRK2+/− T cells. The reduced GRK2 levels in the heterozygote T cells had no significant effect on migration to CCL19 or CCL21 compared to WT T cells. Titration transfection experiments in HEK293T cells or CRISPR-Cas9 knockdown of GRK2 in CEM T cells suggested that GRK2 is required for ligand-mediated CCR7 internalization, while higher GRK2 levels inhibit the internalization process. CCR7 internalization was blocked following transfection of a kinase dead GRK2 mutant indicating that GRK2 phosphorylation is required for CCR7 internalization. Immunomicroscopy suggested arrestin-3 formed clusters, independent of the ligand used. Overall, these data suggest that cellular concentration of GRK2 plays an important role in the internalization of ligand/CCR7.

GPCR Kinase (GRK)-2 Is a Key Negative Regulator of Itch: l-Glutamine Attenuates Itch via a Rapid Induction of GRK2 in an ERK-Dependent Way

Many itch mediators activate GPCR and trigger itch via activation of GPCR-mediated signaling pathways. GPCRs are desensitized by GPCR kinases (GRKs). The aim of this study is to explore the role of GRKs in itch response and the link between GRKs and glutamine, an amino acid previously shown to be an itch reliever. Itch responses were evoked by histamine, chloroquine, and dinitrochlorobenzene-induced contact dermatitis (CD). Phosphorylation and protein expression were detected by immunofluorescent staining and Western blotting. GRK2 knockdown using small interfering RNA enhanced itch responses evoked by histamine, chloroquine, and dinitrochlorobenzene-induced CD, whereas GRK2 overexpression using GRK2-expressing adenovirus reduced the itch responses. Glutamine reduced all itch evoked by histamine, chloroquine, and dinitrochlorobenzene-induced CD. Glutamine-mediated inhibition of itch was abolished by GRK2 knockdown. Glutamine application resulted in a rapid and strong expression of GRK2 in not only dinitrochlorobenzene-induced CD (within 10 minutes) but also cultured rat dorsal root ganglion cells, F11 (within 1 minute). ERK inhibitor abrogates glutamine-mediated GRK2 expression and inhibition of itch in dinitrochlorobenzene-induced CD. Our data indicate that GRK2 is a key negative regulator of itch and that glutamine attenuates itch via a rapid induction of GRK2 in an ERK-dependent way.

GRK2 targeted knock-down results in spontaneous hypertension, and altered vascular GPCR signaling.

Hypertension, elevated arterial pressure, occurs as the consequence of increased peripheral resistance. G protein-coupled receptors (GPCRs) contribute to the regulation of vasodilator and vasoconstrictor responses, and their activity is regulated by a family of GPCR kinases (GRKs). GRK2 expression is increased in hypertension and this facilitates the development of the hypertensive state by increasing the desensitization of GPCRs important for vasodilation. We demonstrate here, that genetic knockdown of GRK2 using a small hairpin (sh) RNA results in altered vascular reactivity and the development of hypertension between 8–12 weeks of age in shGRK2 mice due to enhanced Gαq/11 signaling. Vascular smooth muscle cells (VSMCs) cultured from shGRK2 knockdown mice show increases in GPCR-mediated Gαs and Gαq/11 signaling, as the consequence of reduced GRK2-mediated desensitization. In addition, agonists and biased agonists exhibited age-dependent alterations in ERK1/2 and Akt signaling, as well as cell proliferation and migration responses in shGRK2 knockdown VSMCs when cultured from mice that are either 3 months or 6 months of age. Changes in angiotensin II-stimulated ERK1/2 phosphorylation are observed in VSMCs derived from 6-week-old shGRK2 mice prior to the development of the hypertensive phenotype. Thus, our findings indicate that the balance between mechanisms regulating vascular tone are shifted to favor vasoconstriction in the absence of GRK2 expression and that this leads to the age-dependent development of hypertension, as a consequence of global alterations in GPCR signaling. Consequently, therapeutic strategies that target GRK2 activity, not expression, may be more effective for the treatment of hypertension.

G-protein-coupled receptor kinase 2 terminates G-protein-coupled receptor function in steroid hormone 20-hydroxyecdysone signaling.

G-protein-coupled receptors (GPCRs) transmit extracellular signals across the cell membrane. GPCR kinases (GRKs) desensitize GPCR signals in the cell membrane. However, the role and mechanism of GRKs in the desensitization of steroid hormone signaling are unclear. In this study, we propose that GRK2 is phosphorylated by protein kinase C (PKC) in response to induction by the steroid hormone 20-hydroxyecdysone (20E), which determines its translocation to the cell membrane of the lepidopteran Helicoverpa armigera. GRK2 protein expression is increased during the metamorphic stage because of induction by 20E. Knockdown of GRK2 in larvae causes accelerated pupation, an increase in 20E-response gene expression, and advanced apoptosis and metamorphosis. 20E induces translocation of GRK2 from the cytoplasm to the cell membrane via steroid hormone ecdysone-responsive GPCR (ErGPCR-2). GRK2 is phosphorylated by PKC on serine 680 after induction by 20E, which leads to the translocation of GRK2 to the cell membrane. GRK2 interacts with ErGPCR-2. These data indicate that GRK2 terminates the ErGPCR-2 function in 20E signaling in the cell membrane by a negative feedback mechanism.

A KSHV microRNA enhances viral latency and induces angiogenesis by targeting GRK2 to activate the CXCR2/AKT pathway.

Kaposi's sarcoma-associated herpesvirus (KSHV) is the causative agent of Kaposi's sarcoma (KS), primary effusion lymphoma (PEL) and multicentric Castleman's disease (MCD). Most tumor cells in these malignancies are latently infected by KSHV. Thus, viral latency is critical for the development of tumor and induction of tumor-associated angiogenesis. KSHV encodes more than two dozens of miRNAs but their roles in KSHV-induced angiogenesis remains unknown. We have recently shown that miR-K12-3 (miR-K3) promoted cell migration and invasion by targeting GRK2/CXCR2/AKT signaling (PLoS Pathog, 2015;11(9):e1005171). Here, we further demonstrated a role of miR-K3 and its induced signal pathway in KSHV latency and KSHV-induced angiogenesis. We found that overexpression of miR-K3 not only promoted viral latency by inhibiting viral lytic replication, but also induced angiogenesis. Further, knockdown of GRK2 inhibited KSHV replication and enhanced KSHV-induced angiogenesis by enhancing the CXCR2/AKT signals. As a result, blockage of CXCR2 or AKT increased KSHV replication and decreased angiogenesis induced by PEL cells in vivo. Finally, deletion of miR-K3 from viral genome reduced KSHV-induced angiogenesis and increased KSHV replication. These findings indicate that the miR-K3/GRK2/CXCR2/AKT axis plays an essential role in KSHV-induced angiogenesis and promotes KSHV latency, and thus may be a potential therapeutic target of KSHV-associated malignancies.

G Protein‐Coupled Receptor Kinase 2's Effect on TNFα Signaling Modulates Wound Healing in Intestinal Epithelial Cells

G protein‐coupled receptor kinases (GRKs) are serine/threonine protein kinases critical in the regulation of GPCR lifecycle. Recently GRKs have been shown to have a large interactome and can play a critical role in cell signaling pathways as well as in consequent cell physiological processes. In this study, we examined the role that GRK2 in TNFα signaling in colon epithelial cell biological processes including wound healing, proliferation, apoptosis, and gene expression. Using an siRNA based knockdown approach in SW480 cell line, we find that GRK2 mediates rate of wound closure under basal conditions. GRK2 did not affect either apoptosis or proliferation of these cells. Interestingly, upon examining the levels of various gene transcripts, we observed that matrix metalloproteinases (MMP) 7 and 9 as well as urokinase plasminogen activator (uPa) (proteins involved in cell migration) was markedly upregulated in GRK2 knockdown cells. To assess the mechanism by which GRK2 affects these physiological processes, we examined the role of GRK2 in TNFα‐induced MAPK and NFkB pathways. Our results demonstrate opposing roles of GRK2 in MAPK and NFkB pathways‐while GRK2 mediates NFkB pathway, it inhibits ERK pathway. Our results further demonstrate that GRK2‐mediated regulation of MMP9 is via the ERK pathway. Together, these data suggest that GRK2 plays a critical role in cellular migration and/or wound healing by modulating MMP7, 9 and uPA levels potentially via differential regulation of MAPK and NFkB signaling pathways. Our results provide a novel role for GRK2 in TNFa signaling in colon epithelial cells.

Endothelin-1 suppresses insulin-stimulated Akt phosphorylation and glucose uptake via GPCR kinase 2 in skeletal muscle cells.

Endothelin-1 (ET-1) reduces insulin-stimulated glucose uptake in skeletal muscle, inducing insulin resistance. Here, we have determined the molecular mechanisms underlying negative regulation by ET-1 of insulin signalling. We used the rat L6 skeletal muscle cells fully differentiated into myotubes. Changes in the phosphorylation of Akt was assessed by Western blotting. Effects of ET-1 on insulin-stimulated glucose uptake was assessed with [(3) H]-2-deoxy-d-glucose ([(3) H]2-DG). The C-terminus region of GPCR kinase 2 (GRK2-ct), a dominant negative GRK2, was overexpressed in L6 cells using adenovirus-mediated gene transfer. GRK2 expression was suppressed by transfection of the corresponding short-interfering RNA (siRNA). In L6 myotubes, insulin elicited sustained Akt phosphorylation at Thr(308) and Ser(473) , which was suppressed by ET-1. The inhibitory effects of ET-1 were prevented by treatment with a selective ETA receptor antagonist and a Gq protein inhibitor, overexpression of GRK2-ct and knockdown of GRK2. Insulin increased [(3) H]2-DG uptake rate in a concentration-dependent manner. ET-1 noncompetitively antagonized insulin-stimulated [(3) H]2-DG uptake. Blockade of ETA receptors, overexpression of GRK2-ct and knockdown of GRK2 prevented the ET-1-induced suppression of insulin-stimulated [(3) H]2-DG uptake. In L6 myotubes overexpressing FLAG-tagged GRK2, ET-1 facilitated the interaction of endogenous Akt with FLAG-GRK2. Activation of ETA receptors with ET-1 suppressed insulin-induced Akt phosphorylation at Thr(308) and Ser(473) and [(3) H]2-DG uptake in a GRK2-dependent manner in skeletal muscle cells. These findings suggest that ETA receptors and GRK2 are potential targets for overcoming insulin resistance.

GRK2 Up-Regulation Creates a Positive Feedback Loop for Catecholamine Production in Chromaffin Cells.

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.

Role of G Protein‐Coupled Receptor Kinase 2 in TNFα Signaling in Colon Epithelial Cells

G protein-coupled receptor kinases (GRKs) compose a 7-member family of serine/threonine protein kinases that are critical in the regulation of GPCR lifecycle. In addition to GPCR phosphorylation, GRKs have been shown to have a large interactome and can play a critical role in cell signaling pathways as well as in consequent cell physiological processes. We wanted to test the hypothesis that GRK2 plays an essential role in colon epithelial cell biology in response to TNFα. For this we used colon epithelial cell line (SW480) that were transfected with either control or GRK2 siRNA. The cells were treated with TNFα and assessed for various cell physiological processes including wound healing, proliferation, apoptosis, and gene expression. Our results demonstrate that GRK2 has a positive role basally in the rate of wound closure although the addition of TNFα restored that rate in the siGRK2 group to control levels. Interestingly, upon examining the levels of various gene transcripts, we observed that matrix metalloproteinases (MMP) 7 and 9 as well as urokinase plasminogen activator (uPa) (proteins involved in cell migration) were markedly upregulated in GRK2 knockdown cells. To assess the mechanism by which GRK2 affects these physiological processes, we examined MAPK and NFkB pathways following TNFα treatment. We found that knockdown of GRK2 significantly inhibits TNFa-induced IkBa phosphorylation whereas ERK2 phosphorylation was significantly enhanced. These data suggest that GRK2 may play a critical role in cellular migration and/or wound healing by modulating MMP7, 9 and uPA levels potentially via differential regulation of MAPK and NFkB signaling pathways. Together, our results provide a novel role for GRK2 in TNFa signaling in colon epithelial cells. Supported by NIH Grant.

G-protein-coupled receptor kinase 2-mediated desensitization of adiponectin receptor 1 in failing heart.

Phosphorylative desensitization of G-protein-coupled receptors contributes significantly to post-myocardial infarction (MI) remodeling and heart failure (HF). Here, we determined whether adiponectin receptors (AdipoRs) 1 and 2 (the 7-transmembrane domain-containing receptors mediating adiponectin functions) are phosphorylatively modified and functionally impaired after MI. Post-MI HF was induced by coronary artery occlusion. Receptor phosphorylation, kinase expression, and adiponectin function were determined via in vivo, ex vivo, and in vitro models. AdipoR1 and AdipoR2 are not phosphorylated in the normal heart. However, AdipoR1 was significantly phosphorylated after MI, peaking at 7 days and remaining significantly phosphorylated thereafter. The extent of post-MI AdipoR1 phosphorylation positively correlated with the expression level of GPCR kinase (GRK) 2, the predominant GRK isoform upregulated in the failing heart. Cardiac-specific GRK2 knockout virtually abolished post-MI AdipoR1 phosphorylation, whereas virus-mediated GRK2 overexpression significantly phosphorylated AdipoR1 and blocked adiponectin metabolic-regulatory/anti-inflammatory signaling. Mass spectrometry identified serine-7, threonine-24, and threonine-53 (residues located in the n-terminal intracellular AdipoR1 region) as the GRK2 phosphorylation sites. Ex vivo experiments demonstrated that adenosine monophosphate-activated protein kinase activation and the anti-tumor necrosis factor-α effect of adiponectin were significantly inhibited in cardiomyocytes isolated from nonischemic area 7 days after MI. In vivo experiments demonstrated that acute adiponectin administration-induced cardiac GLUT4 translocation and endothelial nitric oxide synthase phosphorylation were blunted 7 days after MI. Continuous adiponectin administration beginning 7 days after MI failed to protect the heart from adverse remodeling and HF progression. Finally, cardiac-specific GRK2 knockdown restored the cardioprotective effect of adiponectin. AdipoR1 is phosphorylatively modified and desensitized by GRK2 in failing cardiomyocytes, contributing to post-MI remodeling and HF progression.

GRK2 Targeted Knock-down Results in Spontaneous Hypertension, and Altered Vascular GPCR Signaling

Hypertension, elevated arterial pressure, occurs as the consequence of increased peripheral resistance. G protein-coupled receptors (GPCRs) contribute to the regulation of vasodilator and vasoconstrictor responses, and their activity is regulated by a family of GPCR kinases (GRKs). GRK2 expression is increased in hypertension and this facilitates the development of the hypertensive state by increasing the desensitization of GPCRs important for vasodilation. We demonstrate here, that genetic knockdown of GRK2 using a small hairpin (sh) RNA results in altered vascular reactivity and the development of hypertension between 8-12 weeks of age in shGRK2 mice due to enhanced Gαq/11 signaling. Vascular smooth muscle cells (VSMCs) cultured from shGRK2 knockdown mice show increases in GPCR-mediated Gαs and Gαq/11 signaling, as the consequence of reduced GRK2-mediated desensitization. In addition, agonists and biased agonists exhibited age-dependent alterations in ERK1/2 and Akt signaling, as well as cell proliferation and migration responses in shGRK2 knockdown VSMCs when cultured from mice that are either 3 months or 6 months of age. Changes in angiotensin II-stimulated ERK1/2 phosphorylation are observed in VSMCs derived from 6-week-old shGRK2 mice prior to the development of the hypertensive phenotype. Thus, our findings indicate that the balance between mechanisms regulating vascular tone are shifted to favor vasoconstriction in the absence of GRK2 expression and that this leads to the age-dependent development of hypertension, as a consequence of global alterations in GPCR signaling. Consequently, therapeutic strategies that target GRK2 activity, not expression, may be more effective for the treatment of hypertension.

Regulation of FcϵRI Signaling in Mast Cells by G Protein-coupled Receptor Kinase 2 and Its RH Domain

Agonist-induced phosphorylation of G protein-coupled receptors (GPCRs) by GPCRkinases (GRKs) promotes their desensitization and internalization. Here, we sought to determine the role of GRK2 on Fc∈RI signaling and mediator release in mast cells. The strategies utilized included lentiviral shRNA-mediated GRK2 knockdown, GRK2 gene deletion (GRK2(flox/flox)/cre recombinase) and overexpression of GRK2 and its regulator of G protein signaling homology (RH) domain (GRK2-RH). We found that silencing GRK2 expression caused ~50% decrease in antigen-induced Ca(2+) mobilization and degranulation but resulted in ablation of cytokine (IL-6 and IL-13) generation. The effect of GRK2 on cytokine generation does not require its catalytic activity but is mediated via the phosphorylation of p38 and Akt. Overexpression of GRK2 or its RH domain (GRK2-RH) enhanced antigen-induced mast cell degranulation and cytokine generation without affecting the expression levels of any of the Fc∈RI subunits (α, β, and γ). GRK2 or GRK2-RH had no effect on antigen-induced phosphorylation of Fc∈RIγ or Src but enhanced tyrosine phosphorylation of Syk. These data demonstrate that GRK2 modulates Fc∈RI signaling in mast cells via at least two mechanisms.One involves GRK2-RH and modulates tyrosine phosphorylation of Syk, and the other is mediated via the phosphorylation of p38 and Akt.

G protein–coupled receptor kinase 2 (GRK2) is localized to centrosomes and mediates epidermal growth factor–promoted centrosomal separation

G protein-coupled receptor kinases (GRKs) play a central role in regulating receptor signaling, but recent studies suggest a broader role in modulating normal cellular functions. For example, GRK5 has been shown to localize to centrosomes and regulate microtubule nucleation and cell cycle progression. Here we demonstrate that GRK2 is also localized to centrosomes, although it has no role in centrosome duplication or microtubule nucleation. Of interest, knockdown of GRK2 inhibits epidermal growth factor receptor (EGFR)-mediated separation of duplicated centrosomes. This EGFR/GRK2-mediated process depends on the protein kinases mammalian STE20-like kinase 2 (Mst2) and Nek2A but does not involve polo-like kinase 1. In vitro analysis and dominant-negative approaches reveal that GRK2 directly phosphorylates and activates Mst2. Collectively these findings demonstrate that GRK2 is localized to centrosomes and plays a central role in mitogen-promoted centrosome separation most likely via its ability to phosphorylate Mst2.

GRK2 negatively regulates IGF‐1R signaling pathway and cyclins' expression in HepG2 cells

G protein coupled receptor kinase 2 (GRK2) plays a central role in the regulation of a variety of important signaling pathways. Alternation of GRK2 protein level and activity casts profound effects on cell physiological functions and causes diseases such as heart failure, rheumatoid arthritis, and obesity. We have previously reported that overexpression of GRK2 has an inhibitory role in cancer cell growth. To further examine the role of GRK2 in cancer, in this study, we investigated the effects of reduced protein level of GRK2 on insulin-like growth factor 1 receptor (IGF-1R) signaling pathway in human hepatocellular carcinoma (HCC) HepG2 cells. We created a GRK2 knockdown cell line using a lentiviral vector mediated expression of GRK2 specific short hairpin RNA (shRNA). Under IGF-1 stimulation, HepG2 cells with reduced level of GRK2 showed elevated total IGF-1R protein expression as well as tyrosine phosphorylation of receptor. In addition, HepG2 cells with reduced level of GRK2 also demonstrated increased tyrosine phosphorylation of IRS1 at the residue 612 and increased phosphorylation of Akt, indicating a stronger activation of IGF-1R signaling pathway. However, HepG2 cells with reduced level of GRK2 did not display any growth advantage in culture as compared with the scramble control cells. We further detected that reduced level of GRK2 induced a small cell cycle arrest at G2/M phase by enhancing the expression of cyclin A, B1, and E. Our results indicate that GRK2 has contrasting roles on HepG2 cell growth by negatively regulating the IGF-1R signaling pathway and cyclins' expression.