M2 macrophages-derived exosomal miR-3917 promotes the progression of lung cancer via targeting GRK6.

Macrophages in the tumor microenvironment (TME) can serve as potential targets for therapeutic intervention. The aim of this study was to investigate the molecular mechanism by which M2 macrophage-derived exosomes (M2-Ex) affect lung cancer progression through miRNA transport. The THP-1 cells were differentiated into M0 and M2 macrophages. M2-Ex were isolated and identified by transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA). Cancer tissues and adjacent tissues of non-small-cell lung cancer (NSCLC) patients were collected. H1299 and A549 cells were co-cultured with M2-Ex. Subcutaneous xenograft mouse model was established. miR-3917 is highly expressed in lung cancer tissues and M2-Ex. Interference of miR-3917 in M2-Ex inhibits H1299 cell proliferation, migration and invasion, while overexpression of miR-3917 had the opposite effect in A549 cells. M2-Ex promote tumor growth by delivering miR-3917 in vivo. miR-3917 could target G protein-coupled receptor kinase 6 (GRK6), and interference of miR-3917 in M2-Ex inhibits H1299 cells proliferation, migration and invasion by up-regulating GRK6 level, while overexpression of miR-3917 had the opposite effect in A549 cells. M2-Ex can transfer miR-3917 into lung cancer cells and promote lung cancer progression, providing theoretical basis for the diagnosis and effective treatment of lung cancer.

miR-19a may function as a biomarker of oral squamous cell carcinoma (OSCC) by regulating the signaling pathway of miR-19a/GRK6/GPCRs/PKC in a Chinese population.

BACKGROUND: In this study, we aimed to investigate the potential of miR-19a as a biomarker of OSCC and its underlying molecular mechanisms. METHODS: We collected serum and saliva samples from 66 OSCC patients and 66 healthy control subjects. Real-time PCR analysis, bioinformatic analysis and luciferase assays were performed to establish a potential signaling pathway of miR-19a/GRK6/GPCRs/PKC. Flowcytometry and Transwell assays were performed to observe the changes in cell apoptosis, metastasis and invasion. RESULTS: We found that miR-19a, GPR39 mRNA and PKC mRNA were upregulated while GRK6 mRNA was downregulated in the serum and saliva samples collected from OSCC patients. Moreover, in silico analysis confirmed a potential binding site of miR-19a on the 3'UTR of GRK6 mRNA, and the subsequent luciferase assays confirmed the molecular binding between GRK6 and miR-19a. We further identified that the over-expression of miR-19a could regulate the signaling between GRK6, GPR39 and PKC via the signaling pathway of miR-19a/GRK6/GPR39/PKC, which accordingly resulted in suppressed cell apoptosis and promoted cell migration and invasion. CONCLUSION: Collectively, the findings of our study propose that miR-19a is a crucial mediator in the advancement of OSCC, offering a potential avenue for the development of innovative therapeutic interventions aimed at regulating GRK6 and its downstream signaling pathways.

Multisite NHERF1 phosphorylation controls GRK6A regulation of hormone-sensitive phosphate transport.

The type II sodium-dependent phosphate cotransporter (NPT2A) mediates renal phosphate uptake. The NPT2A is regulated by parathyroid hormone (PTH) and fibroblast growth factor 23, which requires Na+/H+ exchange regulatory factor-1 (NHERF1), a multidomain PDZ-containing phosphoprotein. Phosphocycling controls the association between NHERF1 and the NPT2A. Here, we characterize the critical involvement of G protein-coupled receptor kinase 6A (GRK6A) in mediating PTH-sensitive phosphate transport by targeted phosphorylation coupled with NHERF1 conformational rearrangement, which in turn allows phosphorylation at a secondary site. GRK6A, through its carboxy-terminal PDZ recognition motif, binds NHERF1 PDZ1 with greater affinity than PDZ2. However, the association between NHERF1 PDZ2 and GRK6A is necessary for PTH action. Ser162, a PKCα phosphorylation site in PDZ2, regulates the binding affinity between PDZ2 and GRK6A. Substitution of Ser162 with alanine (S162A) blocks the PTH action but does not disrupt the interaction between NHERF1 and the NPT2A. Replacement of Ser162 with aspartic acid (S162D) abrogates the interaction between NHERF1 and the NPT2A and concurrently PTH action. We used amber codon suppression to generate a phosphorylated Ser162(pSer162)-PDZ2 variant. KD values determined by fluorescence anisotropy indicate that incorporation of pSer162 increased the binding affinity to the carboxy terminus of GRK6A 2-fold compared with WT PDZ2. Molecular dynamics simulations predict formation of an electrostatic network between pSer162 and Asp183 of PDZ2 and Arg at position -1 of the GRK6A PDZ-binding motif. Our results suggest that PDZ2 plays a regulatory role in PTH-sensitive NPT2A-mediated phosphate transport and phosphorylation of Ser162 in PDZ2 modulates the interaction with GRK6A.

The Role of G Protein-Coupled Receptor Kinase 6 Regulation in Inflammation and Pain.

The G protein-coupled receptor kinase 6 is associated with inflammation and pathological pain. Impairment of GRK6 expression was described in chronic inflammatory diseases such as rheumatoid arthritis and this was shown to be accompanied by an imbalance of downstream signaling pathways. Here, we discuss novel aspects of GRK6 interaction and its impact upon hyperalgesia and inflammatory processes. In this review, we compile important findings concerning GRK6 regulation for a better pathophysiological understanding of the intracellular interaction in the context of inflammation and show clinical implications-for example, the identification of possible therapy goals in the treatment of chronic inflammatory hyperalgesia.

Suitability of GRK Antibodies for Individual Detection and Quantification of GRK Isoforms in Western Blots.

G protein-coupled receptors (GPCRs) are regulated by GPCR kinases (GRKs) which phosphorylate intracellular domains of the active receptor. This results in the recruitment of arrestins, leading to desensitization and internalization of the GPCR. Aside from acting on GPCRs, GRKs regulate a variety of membrane, cytosolic, and nuclear proteins not only via phosphorylation but also by acting as scaffolding partners. GRKs' versatility is also reflected by their diverse roles in pathological conditions such as cancer, malaria, Parkinson's-, cardiovascular-, and metabolic disease. Reliable tools to study GRKs are the key to specify their role in complex cellular signaling networks. Thus, we examined the specificity of eight commercially available antibodies targeting the four ubiquitously expressed GRKs (GRK2, GRK3, GRK5, and GRK6) in Western blot analysis. We identified one antibody that did not recognize its antigen, as well as antibodies that showed unspecific signals or cross-reactivity. Hence, we strongly recommend testing any antibody with exogenously expressed proteins to clearly confirm identity of the obtained Western blot results. Utilizing the most-suitable antibodies, we established the Western blot-based, cost-effective simple tag-guided analysis of relative protein abundance (STARPA). This method allows comparison of protein levels obtained by immunoblotting with different antibodies. Furthermore, we applied STARPA to determine GRK protein levels in nine commonly used cell lines, revealing differential isoform expression.

Dynamic structure of the full-length scaffolding protein NHERF1 influences signaling complex assembly.

The Na+/H+ exchange regulatory cofactor 1 (NHERF1) protein modulates the assembly and intracellular trafficking of several transmembrane G protein-coupled receptors (GPCRs) and ion transport proteins with the membrane-cytoskeleton adapter protein ezrin. Here, we applied solution NMR and small-angle neutron scattering (SANS) to structurally characterize full-length NHERF1 and disease-associated variants that are implicated in impaired phosphate homeostasis. Using NMR, we mapped the modular architecture of NHERF1, which is composed of two structurally-independent PDZ domains that are connected by a flexible, disordered linker. We observed that the ultra-long and disordered C-terminal tail of NHERF1 has a type 1 PDZ-binding motif that interacts weakly with the proximal, second PDZ domain to form a dynamically autoinhibited structure. Using ensemble-optimized analysis of SANS data, we extracted the molecular size distribution of structures from the extensive conformational space sampled by the flexible chain. Our results revealed that NHERF1 is a diffuse ensemble of variable PDZ domain configurations and a disordered C-terminal tail. The joint NMR/SANS data analyses of three disease variants (L110V, R153Q, and E225K) revealed significant differences in the local PDZ domain structures and in the global conformations compared with the WT protein. Furthermore, we show that the substitutions affect the affinity and kinetics of NHERF1 binding to ezrin and to a C-terminal peptide from G protein-coupled receptor kinase 6A (GRK6A). These findings provide important insight into the modulation of the intrinsic flexibility of NHERF1 by disease-associated point mutations that alter the dynamic assembly of signaling complexes.

Downregulation of GRK6 in arcuate nucleus promotes chronic visceral hypersensitivity via NF-κB upregulation in adult rats with neonatal maternal deprivation.

AIMS: The arcuate nucleus is a vital brain region for coursing of pain command. G protein-coupled kinase 6 (GRK6) accommodates signaling through G protein-coupled receptors. Studies have demonstrated that GRK6 is involved in inflammatory pain and neuropathic pain. The present study was designed to explore the role and the underlying mechanism of GRK6 in arcuate nucleus of chronic visceral pain. METHODS: Chronic visceral pain of rats was induced by neonatal maternal deprivation and evaluated by monitoring the threshold of colorectal distension. Western blotting, immunofluorescence, real-time quantitative polymerase chain reaction techniques, and Nissl staining were employed to determine the expression and mutual effect of GRK6 with nuclear factor κB (NF-κB). RESULTS: Expression of GRK6 in arcuate nucleus was significantly reduced in neonatal maternal deprivation rats when compared with control rats. GRK6 was mainly expressed in arcuate nucleus neurons, but not in astrocytes, and a little in microglial cells. Neonatal maternal deprivation reduced the percentage of GRK6-positive neurons of arcuate nucleus. Overexpression of GRK6 by Lentiviral injection into arcuate nucleus reversed chronic visceral pain in neonatal maternal deprivation rats. Furthermore, the expression of NF-κB in arcuate nucleus was markedly upregulated in neonatal maternal deprivation rats. NF-κB selective inhibitor pyrrolidine dithiocarbamate suppressed chronic visceral pain in neonatal maternal deprivation rats. GRK6 and NF-κB were expressed in the arcuate nucleus neurons. Importantly, overexpression of GRK6 reversed NF-κB expression at the protein level. In contrast, injection of pyrrolidine dithiocarbamate once daily for seven consecutive days did not alter GRK6 expression in arcuate nucleus of neonatal maternal deprivation rats. CONCLUSIONS: Present data suggest that GRK6 might be a pivotal molecule participated in the central mechanisms of chronic visceral pain, which might be mediated by inhibiting NF-κB signal pathway. Overexpression of GRK6 possibly represents a potential strategy for therapy of chronic visceral pain.

Role of GRK6 in the Regulation of Platelet Activation through Selective G Protein-Coupled Receptor (GPCR) Desensitization.

Platelet G protein-coupled receptors (GPCRs) regulate platelet function by mediating the response to various agonists, including adenosine diphosphate (ADP), thromboxane A2, and thrombin. Although GPCR kinases (GRKs) are considered to have the crucial roles in most GPCR functions, little is known regarding the regulation of GPCR signaling and mechanisms of GPCR desensitization by GRKs in platelets. In this study, we investigated the functional role of GRK6 and the molecular basis for regulation of specific GPCR desensitization by GRK6 in platelets. We used GRK6 knockout mice to evaluate the functional role of GRK6 in platelet activation. Platelet aggregation, dense- and -granule secretion, and fibrinogen receptor activation induced by 2-MeSADP, U46619, thrombin, and AYPGKF were significantly potentiated in GRK6-/- platelets compared to the wild-type (WT) platelets. However, collagen-related peptide (CRP)-induced platelet aggregation and secretion were not affected in GRK6-/- platelets. Interestingly, platelet aggregation induced by co-stimulation of serotonin and epinephrine which activate Gq-coupled 5HT2A and Gz-coupled 2A adrenergic receptors, respectively, was not affected in GRK6-/- platelets, suggesting that GRK6 was involved in specific GPCR regulation. In addition, platelet aggregation in response to the second challenge of ADP and AYPGKF was restored in GRK6-/- platelets whereas re-stimulation of the agonist failed to induce aggregation in WT platelets, indicating that GRK6 contributed to P2Y1, P2Y12, and PAR4 receptor desensitization. Furthermore, 2-MeSADP-induced Akt phosphorylation and AYPGKF-induced Akt, extracellular signal-related kinase (ERK), and protein kinase Cδ (PKC) phosphorylation were significantly potentiated in GRK6-/- platelets. Finally, GRK6-/- mice exhibited an enhanced and stable thrombus formation after FeCl3 injury to the carotid artery and shorter tail bleeding times, indicating that GRK6-/- mice were more susceptible to thrombosis and hemostasis. We conclude that GRK6 plays an important role in regulating platelet functional responses and thrombus formation through selective GPCR desensitization.

Age-dependent effects of dopamine receptor inactivation on cocaine-induced behaviors in male rats: Evidence of dorsal striatal D2 receptor supersensitivity.

N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ), which irreversibly inactivates dopamine (DA) receptors, causes pronounced age-dependent behavioral effects in rats. For example, EEDQ either augments or does not affect the DA agonist-induced locomotor activity of preweanling rats while attenuating the locomotion of adolescent and adult rats. The twofold purpose of this study was to determine whether EEDQ would: (a) potentiate or attenuate the cocaine-induced locomotor activity of preweanling, adolescent, and adult rats; and (b) alter the sensitivity of surviving D2 receptors. Rats were treated with vehicle or EEDQ (2.5 or 7.5 mg/kg) on postnatal day (PD) 17, PD 39, and PD 84. In the behavioral experiments, saline- or cocaine-induced locomotion was assessed 24 hr later. In the biochemical experiments, dorsal striatal samples were taken 24 hr after vehicle or EEDQ treatment and later assayed for NPA-stimulated GTPγS receptor binding, G protein-coupled receptor kinase 6 (GRK6), and ß-arrestin-2 (ARRB2). GTPγS binding is a direct measure of ligand-induced G protein activation, while GRK6 and ARRB2 modulate the internalization and desensitization of D2 receptors. Results showed that EEDQ potentiated the locomotor activity of preweanling rats, while attenuating the locomotion of older rats. NPA-stimulated GTPγS binding was elevated in EEDQ-treated preweanling rats, relative to adults, indicating enhanced functional coupling between the G protein and receptor. EEDQ also reduced ARRB2 levels in all age groups, which is indicative of increased D2 receptor sensitivity. In sum, the present results support the hypothesis that D2 receptor supersensitivity is a critical factor mediating the locomotor potentiating effects of EEDQ in cocaine-treated preweanling rats.

MiR-19b-3p attenuates IL-1ß induced extracellular matrix degradation and inflammatory injury in chondrocytes by targeting GRK6.

Osteoarthritis (OA) is characterized by degradation of articular cartilage. MiRNAs are involved in the regulation of chondrogenesis and OA. We aimed to investigate effects and mechanisms of miR-19b-3p in regulating chondrocytes viability, cartilage degradation and inflammatory response. Primary chondrocytes were isolated from cartilages in control subjects and patients with OA. Murine ATDC5 cells were pre-conditioned with IL-1ß in vitro. Expressions and interaction of miR-19b-3p with G protein-coupled receptor kinase 6 (GRK6), and their effects on inflammation, chondrocytes viability and cartilage degradation were determined after miR-19b-3p mimic or GRK6 siRNA transfection. MiR-19b-3p was significantly decreased in OA chondrocytes and IL-1ß-stimulated ATDC5 cells, in paralleled with the elevated type-II-collagen, aggrecan, MMP13 and GRK6 expression. MiR-19b-3p mimic dramatically increased the viability of chondrocytes and suppressed cell apoptosis. It also increased type-II-collagen, aggrecan expression and glycosaminoglycan (sGAG) content, and decreased the expression of MMP-1 and MMP-13 that controlled by IL-1ß. Overexpression of miR-19b-3p inhibited the production of IL-6 and IL-8 in ATDC5 cells. However, the protective effects of miR-19b-3p mimic on IL-1ß induced cell death; IL-8 production and sGAG decrease were greatly discounted by GRK6 lentiviral vectors. Luciferase reporter assay confirmed that GRK6 gene was a direct target ofmiR-19b-3p. GRK6 siRNA transfection antagonized the IL-1ß-induced chondrocytes injury, extracellular matrix degradation and inflammatory response. MiR-19b-3p mimic and GRK6 siRNA showed comparable inhibitory effect on IL-1ß-provoked NF-κB as reflected by the expression of p-p65. NF-κB translocation inhibition with PS1154 reversed the effects of IL-1ß on IL-8 and sGAG. Collectively, miR-19b-3p attenuated OA by targeting GRK6-NF-κB pathway.

Role of G protein-coupled receptor kinase-6 in Escherichia coli lung infection model in mice.

G protein-coupled receptor kinase-6 (GRK6) is a serine/threonine kinase that is important in inflammatory processes. In this study, we examined the role of GRK6 in Escherichia coli-induced lung infection and inflammation using GRK6 knockout (KO) and wild-type (WT) mice. Intratracheal instillation of E. coli significantly enhanced bacterial load in the bronchoalveolar lavage (BAL) of KO compared with WT mice. Reduced bacterial clearance in the KO mice was not due to an intrinsic defect in neutrophil phagocytosis or killing but as a result of reduced neutrophil numbers in the KO BAL. Interestingly, neutrophil numbers in the lung were increased in the KO compared with WT mice, suggesting a potential dysfunction in transepithelial migration of neutrophils from the lungs to the bronchoalveolar space. This effect was selective for lung tissue because peritoneal neutrophil numbers were similar between the two genotypes following peritoneal infection. Although neutrophil expression of CXCR2/CXCR3 was similar between WT and KO, IL-17A expression was higher in the KO compared with WT mice. These results suggest that enhanced neutrophil count in the KO lungs but reduced numbers in BAL are likely due to transepithelial migration defect and/or altered chemokines/cytokines. Together, our studies suggest a previously unrecognized and novel role for GRK6 in neutrophil migration specific to pulmonary tissue during bacterial infection.

The association of single-nucleotide polymorphisms in the oxytocin receptor and G protein-coupled receptor kinase 6 (GRK6) genes with oxytocin dosing requirements and labor outcomes.

BACKGROUND: Oxytocin is a potent uterotonic agent that is widely used for induction and augmentation of labor. Oxytocin has a narrow therapeutic index and the optimal dosing for any individual woman varies widely. OBJECTIVE: The objective of this study was to determine whether genetic variation in the oxytocin receptor (OXTR) or in the gene encoding G protein-coupled receptor kinase 6 (GRK6), which regulates desensitization of the oxytocin receptor, could explain variation in oxytocin dosing and labor outcomes among women being induced near term. STUDY DESIGN: Pregnant women with a singleton gestation residing in Durham County, NC, were prospectively enrolled as part of the Healthy Pregnancy, Healthy Baby cohort study. Those women undergoing an induction of labor at 36 weeks or greater were genotyped for 18 haplotype-tagging single-nucleotide polymorphisms in OXTR and 7 haplotype-tagging single-nucleotide polymorphisms in GRK6 using TaqMan assays. Linear regression was used to examine the relationship between maternal genotype and maximal oxytocin infusion rate, total oxytocin dose received, and duration of labor. Logistic regression was used to test for the association of maternal genotype with mode of delivery. For each outcome, backward selection techniques were utilized to control for important confounding variables and additive genetic models were used. Race/ethnicity was included in all models because of differences in allele frequencies across populations, and Bonferroni correction for multiple testing was used. RESULTS: DNA was available from 482 women undergoing induction of labor at 36 weeks or greater. Eighteen haplotype-tagging single-nucleotide polymorphisms within OXTR and 7 haplotype-tagging single-nucleotide polymorphisms within GRK6 were examined. Five single-nucleotide polymorphisms in OXTR showed nominal significance with maximal infusion rate of oxytocin, and two single-nucleotide polymorphisms in OXTR were associated with total oxytocin dose received. One single-nucleotide polymorphism in OXTR and two single-nucleotide polymorphisms in GRK6 were associated with duration of labor, one of which met the multiple testing threshold (P = .0014, rs2731664 [GRK6], mean duration of labor, 17.7 hours vs 20.2 hours vs 23.5 hours for AA, AC, and CC genotypes, respectively). Three single-nucleotide polymorphisms, two in OXTR and one in GRK6, showed nominal significance with mode of delivery. CONCLUSION: Genetic variation in OXTR and GRK6 is associated with the amount of oxytocin required as well as the duration of labor and risk for cesarean delivery among women undergoing induction of labor near term. With further research, pharmacogenomic approaches may potentially be utilized to develop personalized treatment to improve safety and efficacy outcomes among women undergoing induction of labor.

Overexpression of GRK6 attenuates neuropathic pain via suppression of CXCR2 in rat dorsal root ganglion.

G protein-coupled kinase (GRK) 6 is a member of the GRK family that mediates agonist-induced desensitization and signaling of G protein-coupled receptors (GPCRs), thus involving in a wide variety of processes including inflammation and nociception. Recent studies have indicated that chemokines play an important role in chronic pain via increased expression of respective GPCRs. This study was designed to investigate the role of GRK6 and its interaction with substrate chemokine receptors in dorsal root ganglion (DRG) in a rat model of neuropathic pain induced by chronic constriction injury (CCI). Following induction of CCI, GRK6 expression was significantly downregulated in rat DRGs at L4-L6 segments. Overexpression of GRK6 using lentiviral-mediated production strategy via sciatic nerve injection markedly attenuated mechanical allodynia and thermal hyperalgesia in CCI rats. Overexpression of GRK6 also drastically reversed the hyperexcitability of DRG neurons innervating the hind paw and suppressed the enhanced expression of CXCR2 in DRGs of CCI rats. In addition, co-immunoprecipitation, immunofluorescence, and correlation analysis supported the interaction between GRK6 and CXCR2. These results suggest that GRK6 might be a key molecular involved in peripheral mechanism of neuropathic pain and that overexpression of GRK6 might be a potential strategy for treatment for neuropathic pain through inhibition of CXCR2 signal pathway.

GRK6 palmitoylation increasing its membrance translocation promotes LPS-induced inflammation by PI3K/ AKT pathway in kuppfer cells.

BACKGROUND: G protein-coupled receptor kinases 6 (GRK6) is one kinase of GPCRs, previous studies have shown that GRK6 is involved in the regulation of inflammatory processes. However, the role of GRK6 in inflammation is not well understood and what is the effect of its palmitoylation modification on inflammatory response in macrophage are still largely unknown. METHODS: LPS stimulated Kupffer cells to simulate inflammatory injury model. SiGRK6 and GRK6 lentiviral plasmids were used to alter cellular GRK6 levels. Subcellular localization of GRK6 was detected using Membrane and Cytoplasmic Protein Extraction Kit and immunofluorescence. Palmitoylated Protein Assay Kit (Red) and modified Acyl-RAC method were used to detect palmitoylation levels. RESULTS: GRK6 mRNA and protein expression decreased in LPS-induced inflammatory response in Kupffer cells (P < 0.05). Overexpression of GRK6 promoted inflammatory response, while silencing GRK6 reduced inflammatory response (P < 0.05). In terms of molecular mechanisms, LPS induced increased palmitoylation of GRK6 and promoted the translocation of GRK6 to cell membranes (P < 0.05). Subsequently, GRK6 functioned through the PI3K/ AKT signaling pathway (P < 0.05). Inhibition of palmitoylation level of GRK6 can inhibit its membrane translocation and reduce inflammatory response (P < 0.05). CONCLUSION: Inhibition of palmitoylation level of GRK6 might relieve LPS-induced inflammation in Kupffer cells by blocking GRK6 membrane translocation and subsequent inflammatory signaling pathway, providing a theoretical basis for targeting GRK6 to regulate inflammation.

Overexpression of GRK6 alleviates chronic visceral hypersensitivity through downregulation of P2Y6 receptors in anterior cingulate cortex of rats with prenatal maternal stress.

AIMS: Visceral hypersensitivity is a major clinic symptom in patients with irritable bowel syndrome (IBS). Anterior cingulate cortex (ACC) is involved in processing the information of pain. Both G protein-coupled receptor kinase 6 (GRK6) and P2Y purinoceptor 6 (P2Y6) are associated with neuroinflammation and pathological pain. The aim of this study was to investigate the interaction between GRK6 and P2Y6 in ACC in the development of visceral hypersensitivity of adult offspring rats with prenatal maternal stress (PMS). METHODS: Visceral hypersensitivity was quantified by abdominal withdrawal reflex threshold to colorectal distension (CRD). The expression and cellular distribution of GRK6 and P2Y6 were determined by Western blotting, qPCR, and fluorescence immunohistochemistry. Co-immunoprecipitation was used to evaluate the interaction between GRK6 and P2Y6. RESULTS: The mRNA and protein levels of GRK6 were significantly decreased in ACC of PMS rats. The injection of GRK6 overexpression virus significantly attenuated visceral hypersensitivity of PMS rats. P2Y6's mRNA level, protein level, and ratio of membrane protein over total protein expression was markedly increased in PMS rats. P2Y6 antagonist MRS2578 microinjection reversed visceral hypersensitivity of PMS rats. GRK6 overexpression significantly reduced P2Y6's expression in membrane proteins and P2Y6's ratio of membrane protein over total protein expression. CONCLUSIONS: These results indicate that decreased GRK6 leads to the accumulation of P2Y6 at neuron membrane in ACC, thereby contributing to visceral hypersensitivity of PMS rats.

Nanoluciferase-based complementation assay for systematic profiling of GPCR-GRK interactions.

G protein-coupled receptor kinases (GRKs) are a family of seven soluble receptor-modifying enzymes which are essential regulators of GPCR activity. Following agonist-induced receptor activation and G protein dissociation, GRKs prime the receptor for desensitization through phosphorylation of its C terminus, which subsequently allows arrestins to bind and initiate the receptor internalization process. While GRKs constitute key GPCR-interacting proteins, to date, no method has been put forward to readily and systematically determine the preference of a specific GPCR towards the seven different GRKs (GRK1-7). This chapter describes a simple and standardized approach for systematic profiling of GRK1-7-GPCR interactions relying on the complementation of the split Nanoluciferase (NanoBiT). When applied to a set of GPCRs (MOR, 5-HT1A, B2AR, CXCR3, AVPR2, CGRPR), including two intrinsically ß-arrestin-biased receptors (ACKR2 and ACKR3), this methodology yields highly reproducible results highlighting different GRK recruitment profiles. Using this assay, further characterization of MOR, a crucial target in the development of analgesics, reveals not only its GRK fingerprint but also related kinetics and activity of various ligands for a single GRK.

GRK6 Depletion Induces HIF Activity in Lung Adenocarcinoma.

G protein-coupled receptor kinase 6 (GRK6) is expressed in various tissues and is involved in the development of several diseases including lung cancer. We previously reported that GRK6 is down-regulated in lung adenocarcinoma patients, which induces cell invasion and metastasis. However, further understanding of the role of GRK6 in lung adenocarcinoma is required. Here we explored the functional consequence of GRK6 inhibition in lung epithelial cells. Analysis of TCGA data was coupled with RNA sequencing (RNA-seq) in alveolar epithelial type II (ATII) cells following depletion of GRK6 with RNA interference (RNAi). Findings were validated in ATII cells followed by tissue microarray analysis. Pathway analysis suggested that one of the Hallmark pathways enriched upon GRK6 inhibition is 'Hallmark_Hypoxia' (FDR = 0.014). We demonstrated that GRK6 depletion induces HIF1α (hypoxia-inducible factor 1 alpha) levels and activity in ATII cells. The findings were further confirmed in lung adenocarcinoma samples, in which GRK6 expression levels negatively and positively correlate with HIF1α expression (P = 0.015) and VHL expression (P < 0.0001), respectively. Mechanistically, we showed the impact of GRK6 on HIF activity could be achieved via regulation of VHL levels. Taken together, targeting the HIF pathway may provide new strategies for therapy in GRK6-depleted lung adenocarcinoma patients.

Hypermethylation of the G protein-coupled receptor kinase 6 (GRK6) promoter inhibits binding of C/EBPα, and GRK6 knockdown promotes cell migration and invasion in lung adenocarcinoma cells.

We previously reported that the expression of G protein-coupled receptor kinase 6 (GRK6) is significantly downregulated in lung adenocarcinoma (LADC) tissues, and low expression levels of GRK6 are correlated with poor survival prognosis. However, the specific regulatory mechanisms and functions of GRK6 in LADC remain unknown. Here, we report that GRK6 mRNA expression levels are downregulated in LADC tissues compared to those in matched adjacent non-tumor tissues (P < 0.001). The promoter of the GRK6 gene was found to be hypermethylated in LADC tissues, and its methylation was correlated with both GRK6 expression and pathology grade. GRK6 promoter hypermethylation may predict shorter overall survival. Treatment with 5-aza-2'-deoxycytidine significantly enhanced GRK6 gene expression. Four binding sites of CCAAT/enhancer-binding protein-α (C/EBPα) in the CpG island of the GRK6 gene promoter were predicted in silico, of which three sites were further confirmed by ChIP. Decreased binding of C/EBPα to binding sites 1, 3 and 4 of the GRK6 gene promoter was observed in LADC tissues. Inhibition of C/EBPα significantly inhibited GRK6 expression, while overexpression of C/EBPα significantly promoted GRK6 expression. In addition, overexpression of GRK6 significantly suppressed, while GRK6 knockdown promoted cell migration and invasion. Overexpression of GRK6 enhanced E-cadherin expression and suppressed vimentin expression, and silencing of GRK6 had the opposite effects. Furthermore, ectopic expression of GRK6 significantly decreased matrix metalloproteinase (MMP) 2 and MMP7 protein expression levels. Our findings suggest that hypermethylation of the GRK6 gene promoter suppressed binding of C/EBPα, thereby contributing to the promotion of cell migration and invasion. The methylation status of the GRK6 promoter might be suitable for use as an epigenetic biomarker, and the C/EBPα-GRK6 signaling pathway may be a potential target for LADC.

Down-regulated G protein-coupled receptor kinase 6 leads to apoptosis in multiple myeloma MM1R cells.

G protein-coupled receptor kinase 6 (GRK6) is highly expressed in multiple myeloma (MM) cell lines, but absent or only weakly expressed in most primary human somatic cells. In the present study, GRK6 expression was assessed in MM patients and healthy individuals by quantitative polymerase chain reaction. Flow cytometry were performed to measure the apoptosis of lentivial-transfected MM1R cells. Western blot analysis was performed to assess the apoptosis and signal transducer and activator of transcription 3 pathway-related factors. The results demonstrated that GRK6 was differentially expressed in individuals who suffered from MM and healthy individuals. Previous studies have shown that downregulating GRK6 has anti-cancer effects in the MM cell line, MM1R. The present study demonstrated that RNA interference-mediated GRK6 knockdown promoted apoptosis in the MM1R cell line. Therefore, we hypothesized that GRK6 plays a significant role in determining the course of MM.

G protein-coupled receptor kinase 6 is overexpressed in glioma and promotes glioma cell proliferation.

The expression and potential biological functions of G protein-coupled receptor kinase 6 (GRK6) in human glioma are tested in this study. We show that protein and mRNA expression of GRK6 in human glioma tissues was significantly higher than that in the normal brain tissues. Further immunohistochemistry assay analyzing total 118 human glioma tissues showed that GRK6 over-expression was correlated with glioma pathologic grade and patients' Karnofsky performance status (KPS) score. At the molecular level, in the GRK6-low H4 glioma cells, forced over-expression of GRK6 promoted cell proliferation. Reversely, siRNA-mediated knockdown of GRK6 in the U251MG (GRK6-high) cells led to proliferation inhibition and cell cycle arrest. Intriguingly, GRK6 could also be an important temozolomide resistance factor. Temozolomide-induced cytotoxicity was prominent only in GRK6-low H4 glioma cells. On the other hand, knockdown of GRK6 by targeted siRNA sensitized U251MG cells (GRK6-high) to temozolomide. Thus, GRK6 over-expression in glioma is important for cell proliferation and temozolomide resistance.