Overexpression Of G Protein-coupled Receptor Kinase 2 Research Articles

Abstract 303: Revealing molecular mechanism of G protein-coupled receptor kinase 2 (GRK2)-mediated extracellular matrix remodeling in ovarian cancer

Abstract G protein-independent pathway (β-arrestin dependent signaling cascade) is initiated by activated GPCRs phosphorylated by G protein-coupled receptor kinases (GRKs), triggering multiple signaling pathways. The purpose of this research is to reveal the role of GRK2 on the tumor microenvironment (TME). Through in vitro assays, we showed that GRK2 overexpression led to loss of cell-matrix and cell-cell adhesion. The cell spreading assay further indicated that the loss of focal adhesion (FA) assembly and cell size decreased in GRK2-overexpressing cells. In vivo experiments demonstrated that tumor formation and metastasis were suppressed in xenograft mouse models implanted with GRK2-overexpressing ovarian cancer cells. In addition, label-free imaging detection of the collagen pattern suggested that GRK2 overexpression caused bundle loss but the formation of thicker, twisted collagen fibers in xenograft tumor samples. Together with the transcriptome and proteomic data, we hypothesize that GRK2 suppresses cell motility and metastasis by reducing FA and extracellular matrix (ECM) remodeling in ovarian cancer. This research revised the mechanisms underlying GRK2-mediated ovarian cancer metastasis and suggested the potential to target the GRK2-meditated pathway for cancer suppression. Citation Format: Weng I Lei, Shan Yu, Chi Teng Lei, Zhiming Zhang, Tzu Ming Liu, Wakam Chang, Leo Tsz On Lee. Revealing molecular mechanism of G protein-coupled receptor kinase 2 (GRK2)-mediated extracellular matrix remodeling in ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 303.

GRK2-YAP signaling is implicated in pulmonary arterial hypertension development.

Pulmonary arterial hypertension (PAH) is characterized by excessive proliferation of small pulmonary arterial vascular smooth muscle cells (PASMCs), endothelial dysfunction, and extracellular matrix remodeling. G protein-coupled receptor kinase 2 (GRK2) plays an important role in the maintenance of vascular tone and blood flow. However, the role of GRK2 in the pathogenesis of PAH is unknown. GRK2 levels were detected in lung tissues from healthy people and PAH patients. C57BL/6 mice, vascular smooth muscle cell-specific Grk2 -knockout mice ( Grk2ΔSM22 ), and littermate controls ( Grk2flox/flox ) were grouped into control and hypoxia mice ( n =8). Pulmonary hypertension (PH) was induced by exposure to chronic hypoxia (10%) combined with injection of the SU5416 (cHx/SU). The expression levels of GRK2 and Yes-associated protein (YAP) in pulmonary arteries and PASMCs were detected by Western blotting and immunofluorescence staining. The mRNA expression levels of Grk2 and Yes-associated protein ( YAP ) in PASMCs were quantified with real-time polymerase chain reaction (RT-PCR). Wound-healing assay, 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di-phenytetrazoliumromide (MTT) assay, and 5-Ethynyl-2'-deoxyuridine (EdU) staining were performed to evaluate the proliferation and migration of PASMCs. Meanwhile, the interaction among proteins was detected by immunoprecipitation assays. The expression levels of GRK2 were upregulated in the pulmonary arteries of patients with PAH and the lungs of PH mice. Moreover, cHx/SU-induced PH was attenuated in Grk2ΔSM22 mice compared with littermate controls. The amelioration of PH in Grk2ΔSM22 mice was accompanied by reduced pulmonary vascular remodeling. In vitro study further confirmed that GRK2 knock-down significantly altered hypoxia-induced PASMCs proliferation and migration, whereas this effect was severely intensified by overexpression of GRK2 . We also identified that GRK2 promoted YAP expression and nuclear translocation in PASMCs, resulting in excessive PASMCs proliferation and migration. Furthermore, GRK2 is stabilized by inhibiting phosphorylating GRK2 on Tyr86 and subsequently activating ubiquitylation under hypoxic conditions. Our findings suggest that GRK2 plays a critical role in the pathogenesis of PAH, via regulating YAP expression and nuclear translocation. Therefore, GRK2 serves as a novel therapeutic target for PAH treatment.

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

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

Paroxetine increases delta opioid responsiveness in sensory neurons.

There are currently no Food and Drug Administration (FDA)-approved δ-opioid receptor (DOR)-selective agonists, despite having fewer side effects in rodents and nonhuman primates compared with traditional μ-opioid receptor (MOR) therapeutics (Vanderah, 2010). Targeting peripheral receptors is an attractive strategy to reduce abuse potential. However, peripheral opioid receptors do not readily respond to agonists unless primed by inflammation, which would limit their efficacy in noninflammatory pain patients (Stein et al., 1989). It was recently identified that G-protein-coupled receptor kinase 2 (GRK2) maintains DOR incompetence in noninflamed nociceptors (Brackley et al., 2016, 2017). Here, we report that paroxetine, a selective serotonin reuptake inhibitor (SSRI) and potent GRK2 inhibitor (Thal et al., 2012), reduces chronic GRK2 association with membrane DOR, thereby enhancing peripheral DOR-mediated analgesic competence in the absence of inflammation. Interestingly, paroxetine’s effects on GRK2 in vivo are limited to peripheral tissues in the male rat. The effects of paroxetine on DOR competence are notably antagonized by GRK2 overexpression. This is the first study to suggest that paroxetine induces peripheral DOR analgesic competence through a GRK2-dependent mechanism, improving analgesic efficacy in noninflamed tissue. Because paroxetine targets the protein that governs peripheral opioid receptor responsiveness, and does so in the absence of inflammation, we propose that paroxetine may be suitable as a co-therapy with peripherally-restrictive doses of opioids to improve analgesic efficacy in noninflammatory pain conditions.

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

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

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

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

Investigation of G protein‐coupled receptor kinase 2 (GRK2) regulation of D2 dopamine receptor signaling: effects of GRK2 functional domains and pharmacologic and genetic inhibition of GRK2 activity

The D2 dopamine (DA) receptor (D2R) signals through two major pathways, namely activation of G proteins and recruitment of β‐arrestin. Agonist binding stabilizes a receptor state that activates G proteins and facilitates receptor phosphorylation by one or more members of the G protein‐coupled receptor kinase (GRK) family, and this phosphorylation is known to enhance β‐arrestin recruitment and initiate the process of internalization for many GPCRs. Previously, we found that only GRK2 and GRK3 were involved in D2R phosphorylation in response to agonist stimulation (Namkung et al., JBC: 284: 15038, 2009). In this study, we further investigated the influence of GRK2 on receptor signaling, β‐arrestin recruitment, and internalization of the D2R. Overexpression of GRK2 was found to increase DA‐stimulated β‐arrestin recruitment and receptor internalization, whereas it suppressed D2R‐mediated inhibition of cAMP accumulation. In contrast, a catalytically inactive GRK2 mutant (GRK2‐K220R) did not affect any of these functional outputs suggesting that GRK2 kinase activity is required to influence D2R signaling. A disabling mutation of the GRK2 RGS homology (RH) domain (GRK2‐D110A) behaved similarly as WT GRK2, suggesting that this domain is not required for GRK2’s regulatory effects on the D2R. Finally, overexpression of a GRK2 construct containing a mutation in the pleckstrin homology (PH) domain (GRK2‐R587Q), which negates GRK2 interaction with Gβγ subunits, increased DA‐stimulated β‐arrestin recruitment and receptor internalization (more than observed with WT GRK2), but did not affect D2R regulation of cAMP accumulation, indicating that this GRK2 domain is involved in regulating some, but not all, aspects of D2R signaling. To study D2R regulation in the complete absence of GRK2, we utilized a cell line in which GRK2 expression was knocked out using CRISPR‐Cas9 technology. DA‐stimulated β‐arrestin recruitment and receptor internalization were decreased, but not eliminated, in the GRK2 KO cells suggesting that, while GRK2 may be facilitatory, it is not essential for these processes. In parallel studies we characterized the effects of Compound 101, a dual GRK2/3 inhibitor. Compound 101 was found to negate GRK2 interactions with the D2R and attenuate, but not eliminate, β‐arrestin recruitment and receptor internalization. In contrast, in the GRK2 KO cells, Compound 101 had no effect on the residual DA‐stimulated β‐arrestin recruitment. In conclusion, our data suggest that different GRK2 domains affect specific regulatory pathways of the D2R and that both agonist‐induced β‐arrestin recruitment and receptor internalization can occur in the absence of GRK2 interactions with the D2R.Support or Funding InformationNINDS Intramural Research Program

G protein-coupled receptor kinase 2 regulates mitochondrial bioenergetics and impairs myostatin-mediated autophagy in muscle cells.

G protein-coupled receptor kinase 2 (GRK2) is an important protein involved in β-adrenergic receptor desensitization. In addition, studies have shown GRK2 can modulate different metabolic processes in the cell. For instance, GRK2 has been recently shown to promote mitochondrial biogenesis and increase ATP production. However, the role of GRK2 in skeletal muscle and the signaling mechanisms that regulate GRK2 remain poorly understood. Myostatin is a well-known myokine that has been shown to impair mitochondria function. Here, we have assessed the role of myostatin in regulating GRK2 and the subsequent downstream effect of myostatin regulation of GRK2 on mitochondrial respiration in skeletal muscle. Myostatin treatment promoted the loss of GRK2 protein in myoblasts and myotubes in a time- and dose-dependent manner, which we suggest was through enhanced ubiquitin-mediated protein loss, as treatment with proteasome inhibitors partially rescued myostatin-mediated loss of GRK2 protein. To evaluate the effects of GRK2 on mitochondrial respiration, we generated stable myoblast lines that overexpress GRK2. Stable overexpression of GRK2 resulted in increased mitochondrial content and enhanced mitochondrial/oxidative respiration. Interestingly, although overexpression of GRK2 was unable to prevent myostatin-mediated impairment of mitochondrial respiratory function, elevated levels of GRK2 blocked the increased autophagic flux observed following treatment with myostatin. Overall, our data suggest a novel role for GRK2 in regulating mitochondria mass and mitochondrial respiration in skeletal muscle.

Investigating the Interactions of GRK2 with a G‐protein Signaling‐Biased D2 Dopamine Receptor

D2 dopamine receptors (D2Rs) are a part of the G protein‐coupled receptor (GPCR) superfamily, and have been linked to the etiology and/or therapy of numerous neuropsychiatric disorders, such as Parkinson's disease and schizophrenia. While the D2R is a highly validated drug target, the receptor signals through multiple pathways, some of which may result in adverse side effects in patients. Signaling‐biased agents that selectivity stimulate, or block, specific D2R‐mediated pathways could represent improved therapeutics. We recently identified a G‐protein signaling‐biased D2R agonist (MLS1547) that has minimal ability to recruit β‐arrestin. Molecular modeling of MLS1547‐D2R interactions revealed a key residue, F189, at the interface between the fifth transmembrane segment (TM5) and the second extracellular loop that is involved in the biased signaling properties of MLS1547. We mutated this residue in the D2R (F189A) and found, using a variety of signaling assays, that this mutation prevents the recruitment of β‐arrestin with minimal effects on G protein signaling, i.e., the F189A D2R is a G protein signaling‐biased mutant. To investigate the mechanisms of this biased signaling we explored the interactions of the wild‐type D2R and F189 mutant with G‐protein coupled receptor kinase 2 (GRK2), which has been shown to phosphorylate the D2R. Initially, we found that over‐expression of GRK2 can enhance the ability of dopamine to recruit β‐arrestin to the WT receptor. Similarly, using a GRK2‐D2R BRET assay to directly assess D2R‐GRK2 interactions, we found that dopamine stimulation increases GRK2‐D2R interactions. Surprisingly, we found that the F189A D2R mutant is unable to recruit GRK2 upon agonist activation suggesting that GRK2‐D2R interactions may be linked to recruiting β‐arrestin. In another series of experiments, we sought to determine if these effects of GRK2 are mediated by receptor phosphorylation. We examined agonist‐induced β‐arrestin and GRK2 recruitment using previously described phosphorylation‐defective D2R mutants (Namkung et. al., JBC 284:34103, 2009). Interestingly, we found that the D2R phosphorylation‐null mutants were not impaired in their ability to recruit either GRK2 or β‐arrestin – in fact, β‐arrestin recruitment and receptor internalization were actually enhanced. These results suggest that GRK2‐D2R interactions may play a critical role in regulating the biased signaling properties of the D2R, but not through phosphorylation of the receptor. Further, GRK2 may enhance β‐arrestin recruitment by mechanisms other than D2R phosphorylation.Support or Funding InformationNINDS IRPThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Suppression of GRK2 expression reduces endothelial dysfunction by restoring glucose homeostasis

Despite the associations between diabetic complications and vascular endothelial dysfunction, a direct therapeutic method targeting endothelial dysfunction remains poorly characterized. We have previously shown that chemical inhibition of G-protein-coupled receptor kinase 2 (GRK2) slightly enhances insulin sensitivity and reduces endothelial dysfunction in type 2 diabetic mice. In this study, we identified GRK2 as a novel therapeutic target of diabetic endothelial dysfunction and investigated the effect on diabetic endothelial dysfunction through the systemic administration of GRK2 siRNA using a hydrodynamic-based procedure, resulting in suppression of increased GRK2 protein levels in the liver. Suppressed GRK2 levels in the liver markedly improved glucose homeostasis, as well as improved the impaired endothelial Akt/eNOS-dependent signal activation (insulin-stimulated phosphorylation of Akt and eNOS) and vascular responses (clonidine-induced and insulin-induced endothelial-dependent relaxation response and phenylephrine-induced contractile response) in type 2 diabetic aortas. Interestingly, insulin-stimulated Akt/eNOS signaling was increased only by normalizing the glucose concentration in human umbilical vein endothelial cells (HUVECs) with GRK2 overexpression, suggesting of an important role of hepatic GRK2. Our results clarified the relationship among hepatic GRK2, glucose homeostasis, and vascular endothelial function. Liver-targeting GRK2 siRNA delivery represents a novel therapeutic tool to restore glucose homeostasis and reduce endothelial dysfunction.

G protein-coupled receptor kinase 2 promotes cardiac hypertrophy.

The increase in protein activity and upregulation of G-protein coupled receptor kinase 2 (GRK2) is a hallmark of cardiac stress and heart failure. Inhibition of GRK2 improved cardiac function and survival and diminished cardiac remodeling in various animal heart failure models. The aim of the present study was to investigate the effects of GRK2 on cardiac hypertrophy and dissect potential molecular mechanisms. In mice we observed increased GRK2 mRNA and protein levels following transverse aortic constriction (TAC). Conditional GRK2 knockout mice showed attenuated hypertrophic response with preserved ventricular geometry 6 weeks after TAC operation compared to wild-type animals. In isolated neonatal rat ventricular cardiac myocytes stimulation with angiotensin II and phenylephrine enhanced GRK2 expression leading to enhanced signaling via protein kinase B (PKB or Akt), consecutively inhibiting glycogen synthase kinase 3 beta (GSK3β), such promoting nuclear accumulation and activation of nuclear factor of activated T-cells (NFAT). Cardiac myocyte hypertrophy induced by in vitro GRK2 overexpression increased the cytosolic interaction of GRK2 and phosphoinositide 3-kinase γ (PI3Kγ). Moreover, inhibition of PI3Kγ as well as GRK2 knock down prevented Akt activation resulting in halted NFAT activity and reduced cardiac myocyte hypertrophy. Our data show that enhanced GRK2 expression triggers cardiac hypertrophy by GRK2-PI3Kγ mediated Akt phosphorylation and subsequent inactivation of GSK3β, resulting in enhanced NFAT activity.

GRK2 desensitizes flow-induced responses in osteoblasts.

Bone desensitization after mechanical loading is essential for bone to adapt to its mechanical environment. However, the desensitization mechanism is unknown. Previous studies suggest that G protein-coupled receptors (GPCRs), including P2Y and parathyroid hormone receptors, play important roles in osteoblast mechanobiology. Thus, for the present research, we examined the role of G protein-coupled receptor kinase 2 (GRK2) in osteoblast desensitization after exposure to mechanical stimulation. We first showed the existence of osteoblast desensitization after mechanical stimulation based on cytosol Ca2+ and phosphorylated ERK1/2 activities, detected using a fluorescent Ca2+-sensitive dye and western blotting, respectively. We then demonstrated that GRK2 overexpression in MC3T3-E1 cells inhibits flow-induced ERK1/2 phosphorylation, while siRNA knockdown of GRK2 enhances ERK1/2 phosphorylation. Additionally, we found that GRK2 overexpression in MC3T3-E1 cells inhibits cyclooxygenase-2 mRNA expression in the short term and alkaline phosphatase activity in the long term. More importantly, we discovered that GRK2 translocated to the cell membrane shortly after flow stimulation - a step necessary for GPCR desensitization. Previously, we have demonstrated that P2Y2 purinergic receptors, one type of GPCRs, are involved in various flow-induced osteoblastic responses. In this research, we also showed that GRK2 overexpression does not affect ATP release. Accordingly, GRK2 is able to inhibit flow-induced osteoblast responses possibly through desensitizing P2Y2 receptors.

GRK2 Is a Novel Modulator of the MALT1 Oncoprotein

Background and Significance: Lymphocyte antigen receptor-dependent stimulation of the NF-κB transcription factor is a key event during the normal adaptive immune response. However, dysregulated activation of these signaling pathways can lead to lymphoma. Stimulation of the B-or T-cell receptor promotes assembly of the CARMA1-Bcl10-MALT1 (CBM) signaling complex, where MALT1 functions as the essential effector molecule by carrying out two important functions: First, as a scaffold to recruit and activate components of the canonical NF-κB signaling machinery, and second, acts as a protease to enzymatically cleave and inactivate multiple substrates including several negative regulators of canonical NF-κB signaling. Activating mutations in either BCR or CARMA1 promote constitutive and exaggerated MALT1 activity and are seen as driver mutations in diffuse large B cell lymphoma (DLBCL). Aberrant MALT1 activity also underlies the pathogenesis of MALT lymphoma, another subtype of B-cell malignancy. As such, MALT1 has emerged as a potential new target for pharmaceutical inhibition in the treatment of lymphoma. Here, we describe our discovery of a novel MALT1-binding partner, G protein-coupled receptor kinase 2 (GRK2). We hypothesize that GRK2 plays a critical role in the pathogenesis of lymphoma by modulating MALT1 activity.Methods and Results: Using co-immunoprecipitation and liquid chromatography mass spectrometry we identified GRK2 as a novel MALT1-interacting protein. We validated our results by showing that endogenous GRK2 and MALT1 co-immunoprecipitate (CO-IP) in both B cells and T cells. We next utilized GST-tagged purified proteins to demonstrate a direct interaction between GRK2 and MALT1. We then performed a number of studies to investigate the functional consequences of GRK2 interaction with MALT1. A CO-IP assay demonstrated that GRK2 inhibits Bcl10-dependent binding to MALT1, and a luciferase reporter assay revealed that GRK2 inhibits Bcl10/MALT1-dependent NF-κB activity. In addition, we demonstrated that GRK2 also inhibits MALT1 proteolytic activity using Western blot analysis of two known MALT1 substrates, the deubiquitinase cylindromatosis (CYLD) and the NF-kB family member RelB. Moreover, in Jurkat T cells, overexpression of GRK2 impeded AgR-induced NF-κB activation while stable GRK2 knockdown resulted in enhanced AgR-induced phosphorylation of inhibitor of kappaB (IκB) and secretion of interleukin2 (IL-2).We next began to investigate the potential of GRK2 to function as a tumor suppressor by blocking MALT1 activity. Of note, utilizing a published data set to compare GRK2 mRNA expression in 44 DLBCL patient samples to that of 20 samples of healthy B-cell controls, we found that GRK2 mRNA levels are markedly lower in DLBCL cases. We also compared GRK2 mRNA levels among patients diagnosed with DLBCL grouped by vital status, using data from the Cancer Genome Atlas (TCGA). We found that tumors from patients who died of disease within three years expressed significantly lower levels of GRK2 as compared to tumors from patients alive after three years. We also wished to investigate the impact of GRK2 expression on activated B-cell type diffuse large B-cell lymphoma (ABC-DLBCL) cells, which require MALT1 activity for growth and survival. We were successful in engineering GRK2 overexpression in control germinal center B-cell like (GCB) DLBCL cells, which do not depend on MALT1 activity for growth. In contrast, we found that in ABC-DLBCL cells, which do depend on MALT1 activity, GRK2 overexpression greatly impairs cell proliferation, making it difficult to grow these engineered cells. These data provide support for the concept that GRK2 may have a potential tumor suppressor role in MALT1-dependent lymphoma.Conclusions: Our study represents the first report of a negative modulator of MALT1 and provides critical new insight into mechanisms regulating its activity. We hope to harness this information in developing novel strategies for inhibiting MALT1-dependent lymphomagenesis. DisclosuresNo relevant conflicts of interest to declare.

Hypothesis: Paroxetine, a G Protein-Coupled Receptor Kinase 2 (GRK2) Inhibitor Reduces Morbidity and Mortality in Patients With Heart Failure.

The hypothesis that paroxetine decreases morbidity and mortality in patients with heart failure (HF) is plausible but unproven. Basic research demonstrates that inhibition of G protein-coupled receptor kinase 2 (GRK2) both in vitro and in vivo in the myocardium may be beneficial. G protein-coupled receptor kinase 2 antagonism is purported to exert cardioprotective effects immediately following myocardial injury by blunting toxic overstimulation on a recently injured heart. In addition, chronic overexpression of GRK2 inhibits catecholamine induction of vital positive chronotropic and ionotropic effects required to preserve cardiac output leading to worsening of congestive HF. In cardiac-specific GRK2 conditional knockout mice, there is significant improvement in left ventricular wall thickness, left ventricular end-diastolic diameter (LVEDD), and ejection fraction (EF) compared to controls. Paroxetine is a selective serotonin reuptake inhibitor which was recently shown to have the ability to directly inhibit GRK2 both in vitro and in vivo. At physiologic temperatures, paroxetine inhibits GRK2-dependent phosphorylation of an activated G-protein-coupled receptor with a half maximal inhibitory concentration of 35 micromoles, a substantially greater affinity than for other G protein-coupled receptor kinases. In a randomized trial in mice with systolic HF and depressed EF postmyocardial infarction, those treated with paroxetine had a 30% increase in EF, improved contractility, and LVEDD and wall thickness compared to those treated with medical therapy alone. While further basic research may continue to elucidate plausible mechanisms of benefit and observational studies will contribute important relevant information, large scale randomized trials designed a priori to do so are necessary to test the hypothesis.

The effect of G protein-coupled receptor kinase 2 (GRK2) on lactation and on proliferation of mammary epithelial cells from dairy cows

Milk protein is an important component of milk and a nutritional source for human consumption. To better understand the molecular events underlying synthesis of milk proteins, the global gene expression patterns in mammary glands of dairy cow with high-quality milk (>3% milk protein; >3.5% milk fat) and low-quality milk (<3% milk protein; <3.5% milk fat) were examined via digital gene expression study. A total of 139 upregulated and 66 downregulated genes were detected in the mammary tissues of lactating cows with high-quality milk compared with the tissues of cows with low-quality milk. A pathway enrichment study of these genes revealed that the top 5 pathways that were differentially affected in the tissues of cows with high- versus low-quality milk involved metabolic pathways, cancer, cytokine-cytokine receptor interactions, regulation of the actin cytoskeleton, and insulin signaling. We also found that the G protein-coupled receptor kinase 2 (GRK2) was one of the most highly upregulated genes in lactating mammary tissue with low-quality milk compared with tissue with high-quality milk. The knockdown of GRK2 in cultured bovine mammary epithelial cells enhanced CSN2 expression and activated signaling molecules related to translation, including protein kinase B, mammalian target of rapamycin, and p70 ribosomal protein S6 kinase 1 (S6K1), whereas overexpression of GRK2 had the opposite effects. However, expression of genes involved in the mitogen-activated protein kinase pathway was positively regulated by GRK2. Therefore, GRK2 seems to act as a negative mediator of milk-protein synthesis via the protein kinase B-mammalian target of rapamycin signaling axis. Furthermore, GRK2 may negatively control milk-protein synthesis by activating the mitogen-activated protein kinase pathway in dairy cow mammary epithelial cells.

GRK2 overexpression inhibits IGF1-induced proliferation and migration of human hepatocellular carcinoma cells by downregulating EGR1

G protein-coupled receptor kinase 2 (GRK2) is a serine/threonine kinase that is involved in a variety of important signaling pathways and alternation of GRK2 protein level or activity causes diseases such as heart failure, rheumatoid arthritis, and obesity. However, the role and mechanism of GRK2 in hepatocellular carcinoma (HCC) progression is not fully investigated. In this study we found that GRK2 plays an inhibitory role in IGF1-induced HCC cell proliferation and migration. Overexpression of GRK2 causes a decrease in early growth response-1 (EGR1) expression, while knockdown of GRK2 leads to marked increase in EGR1 expression in the treatment of IGF1. Through co-immunoprecipitation and western blot assay, we confirmed that GRK2 can interact with insulin-like growth factor 1 receptor (IGF-1R) and inhibits IGF1-induced activation of IGF1R signaling pathway. Silencing EGR1 attenuates GRK2 overexpression-caused inhibition of cell proliferation, tumor colony number and migration activity, while overexpressing of EGR1 restores the anti-proliferative and migratory effect by GRK2 overexpression in HCCLM3 cells. Collectively, these results suggest that GRK2 may inhibit IGF1-induced HCC cell growth and migration through downregulation of EGR1 and indicate that enforced GRK2 may offer a potential therapeutic approach against HCC.

GRK2 compromises cardiomyocyte mitochondrial function by diminishing fatty acid-mediated oxygen consumption and increasing superoxide levels

The G protein-coupled receptor kinase-2 (GRK2) is upregulated in the injured heart and contributes to heart failure pathogenesis. GRK2 was recently shown to associate with mitochondria but its functional impact in myocytes due to this localization is unclear. This study was undertaken to determine the effect of elevated GRK2 on mitochondrial respiration in cardiomyocytes. Sub-fractionation of purified cardiac mitochondria revealed that basally GRK2 is found in multiple compartments. Overexpression of GRK2 in mouse cardiomyocytes resulted in an increased amount of mitochondrial-based superoxide. Inhibition of GRK2 increased oxygen consumption rates and ATP production. Moreover, fatty acid oxidation was found to be significantly impaired when GRK2 was elevated and was dependent on the catalytic activity and mitochondrial localization of this kinase. Our study shows that independent of cardiac injury, GRK2 is localized in the mitochondria and its kinase activity negatively impacts the function of this organelle by increasing superoxide levels and altering substrate utilization for energy production.

Phosphatidylethanolamine‐Binding Protein Regulates Mu Opioid Receptor Induced βarrestin2 Recruitment and Downstream Signaling

The signaling regulators and pathways of the mu‐opioid receptor (MOR) are being studied to develop functionally selective MOR ligands. Such novel analgesic drugs may alleviate pain without the side effects and drawbacks of typical opioids. We have analyzed proteomics databases available in the literature to find candidate proteins that are regulated in the brain by chronic opioid treatment. One such candidate, phosphatidylethanolamine‐binding protein (PEBP), is a cytoplasmic protein with a highly conserved structure that has been shown to regulate GPCR signaling through both the Raf/MEK/ERK pathway and G protein‐coupled receptor kinase 2 (GRK2), a major feedback inhibitor of GPCRs which has been implicated in a number of neurological disorders including chronic pain. The expression of PEBP was also found to be regulated by chronic morphine treatment in a proteomic study of rodent brain, suggesting that PEBP may regulate MOR signaling, however PEBP has not yet been associated with MOR signaling. We have knocked down PEBP in MOR‐ expressing HEK, U2OS, N2a and SH‐SY5Y cells using specific siRNA to determine changes in either MOR‐induced ERK signaling or βarrestin2 (βarr2) recruitment and receptor internalization, with and without GRK2 overexpression. PEBP knockdown does not change MOR agonist induced ERK signaling efficacy or potency, but βarr2 recruitment, MOR internalization, and chronic morphine desensitization potency is increased. This suggests a role for PEBP in regulating MOR induced βarr2 recruitment and attendant opioid induced side effects. We will continue to study the role of PEBP in pain and opioid response in vivo, and are developing in vitro drug discovery screens with PEBP as a target.

Skeletal muscle myogenesis is regulated by G protein-coupled receptor kinase 2.

G protein-coupled receptor kinase 2 (GRK2) is an important serine/threonine-kinase regulating different membrane receptors and intracellular proteins. Attenuation of Drosophila Gprk2 in embryos or adult flies induced a defective differentiation of somatic muscles, loss of fibers, and a flightless phenotype. In vertebrates, GRK2 hemizygous mice contained less but more hypertrophied skeletal muscle fibers than wild-type littermates. In C2C12 myoblasts, overexpression of a GRK2 kinase-deficient mutant (K220R) caused precocious differentiation of cells into immature myotubes, which were wider in size and contained more fused nuclei, while GRK2 overexpression blunted differentiation. Moreover, p38MAPK and Akt pathways were activated at an earlier stage and to a greater extent in K220R-expressing cells or upon kinase downregulation, while the activation of both kinases was impaired in GRK2-overexpressing cells. The impaired differentiation and fewer fusion events promoted by enhanced GRK2 levels were recapitulated by a p38MAPK mutant, which was able to mimic the inhibitory phosphorylation of p38MAPK by GRK2, whereas the blunted differentiation observed in GRK2-expressing clones was rescued in the presence of a constitutively active upstream stimulator of the p38MAPK pathway. These results suggest that balanced GRK2 function is necessary for a timely and complete myogenic process.

Phosphorylation of Epac1 by GRK2 inhibits Epac1‐Rap1 signaling and prevents chronic pain (802.7)

Chronic pain is a major health issue that severely affects the quality of life of patients and it financially burdens the society. The understanding of molecular mechanisms that cause transition from acute to chronic pain remains a challenge and is critical for development of new interventions. G‐protein coupled receptor Kinase 2 (GRK2) is a key molecule in pain signaling. We recently described that GRK2 deficiency in nociceptors promotes transition to chronic inflammatory pain via an Exchange protein directly activated by cAMP (Epac) dependent pathway. The objective of this study was to investigate the mechanism via which GRK2 regulates cAMP induced Epac1 signaling. Using co‐immunoprecipitation studies we show that GRK2 binds Epac1 in neuronal cells. Epac1 specifically interacts with the kinase domain of GRK2. In vitro kinase assays revealed that GRK2 is an Epac1 kinase. Overexpression of GRK2, but not of GRK2 kinase dead mutant significantly reduced Epac‐to‐Rap1 signaling. Furthermore, we identified the Epac1 residue that is phosphorylated by GRK2 and this phosphorylation inhibits Epac1‐to‐Rap1 signaling. We conclude that GRK2 acts as an endogenous inhibitor of Epac1‐to‐Rap1 signaling by phosphorylating Epac1. In vivo, GRK2 kinase activity is required to inhibit transition to chronic pain. Thus, we uncovered a novel regulatory mechanism that is critical for regulating the pain response.