G Protein-coupled Receptor Kinase 6 Expression Research Articles

Therapeutic potential and protective role of GRK6 overexpression in pulmonary arterial hypertension

Abnormal proliferation of pulmonary arterial smooth muscle cells (PASMCs) is a key mechanism in the development of pulmonary arterial hypertension (PAH). Signal transducer and activator of transcription 3 (STAT3) signalling plays a critical role in modulating PASMC proliferation, and G-protein-coupled receptor kinase 6 (GRK6) regulates the STAT3 pathway. However, the mechanism underlying the relationship between GRK6 and PAH remains unclear. In this study, we aimed to investigate the role of GRK6 in PAH and determine its potential as a therapeutic target. We utilised hypoxia- and SU5416-induced PAH mouse models and a monocrotaline-induced PAH rat model to analyse the involvement of GRK6. We conducted gain- and loss-of-function experiments using mouse PASMCs. Modulation of GRK6 expression was achieved via a lentiviral vector in vitro and an adeno-associated virus serotype 1 encoding GRK6 in vivo. GRK6 was significantly downregulated in the lung tissues of PAH mice and rats, predominantly in PASMCs. Knockout of GRK6 exacerbated PAH, while both therapeutic and prophylactic overexpression of GRK6 alleviated PAH, as evidenced by a reduction in right ventricular systolic pressure, right ventricular wall to left ventricular wall plus ventricular septum ratio, pulmonary vascular media thickness, and pulmonary vascular muscularisation. Mechanistically, GRK6 overexpression attenuated hypoxia-induced PASMC proliferation and STAT3 phosphorylation. Conversely, knockdown of GRK6 promoted hypoxia-induced proliferation, which was mitigated by a STAT3 inhibitor. Our findings highlight the potential protective and beneficial roles of GRK6 in PAH; we propose a lung-targeted GRK6 gene therapy utilizing adeno-associated virus serotype 1 as a potential treatment approach for patients with PAH.

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

AimsVisceral 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).MethodsVisceral 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.ResultsThe 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.ConclusionsThese results indicate that decreased GRK6 leads to the accumulation of P2Y6 at neuron membrane in ACC, thereby contributing to visceral hypersensitivity of PMS rats.

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.

MiR-19b-3p attenuates IL-1β 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-1β 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-1β-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-1β. 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-1β 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-1β-induced chondrocytes injury, extracellular matrix degradation and inflammatory response. MiR-19b-3p mimic and GRK6 siRNA showed comparable inhibitory effect on IL-1β-provoked NF-κB as reflected by the expression of p-p65. NF-κB translocation inhibition with PS1154 reversed the effects of IL-1β on IL-8 and sGAG. Collectively, miR-19b-3p attenuated OA by targeting GRK6-NF-κB pathway.

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.

Overexpression of G Protein-Coupled Receptor Kinase 6 (GRK6) Is Associated with Progression and Poor Prognosis of Papillary Thyroid Carcinoma.

BackgroundApproximately 20% of patients with papillary thyroid carcinoma (PTC) will develop cancer recurrence, but no clinically available biomarker has been identified. Our study aimed to evaluate the prognostic value of G protein-coupled receptor kinase 6 (GRK6) in PTCs.Material/MethodsWe retrospectively enrolled 108 PTC patients in this study, and explored the expression of GRK6 in resected tumor samples by RT-qPCR and immunohistochemistry (IHC). The clinical data were interpreted by chi-square test, univariate analysis, and multivariate analysis. To investigate the functional mechanisms of GRK6 in regulating PTC progression, we also performed overexpression and silencing experiments in TPC-1 cells, a cell line generated from PTC tissues.ResultsRT-qPCR results showed a higher level of GRK6-mRNA in PTCs than in adjacent thyroid tissues. IHC revealed a distinct protein expression pattern of GRK6 among PTCs. Accordingly, we classified patients into low-GRK6 and high-GRK6 groups. The chi-square test showed that a higher GRK6 was associated with larger tumor size (P=0.045) and advanced TNM stage (P=0.001). Kaplan-Meier survival curve and log rank test demonstrated that higher GRK6 predicted poor disease-free survival (DFS) in PTC patients (P=0.002). Furthermore, Cox regression analysis confirmed that GRK6 was an independent prognostic factor for a higher recurrence risk of PTCs (P=0.047). MTT assay and Transwell assay demonstrated that GRK6 overexpression can significantly enhance tumor proliferation and invasion, which was consistent with clinical findings.ConclusionsOur data show the oncogenic effects of GRK6 in promoting PTC progression.

Overexpression of GRK6 associates with the progression and prognosis of colorectal carcinoma.

There are a limited number of studies reporting on the expression of G protein-coupled receptor kinase 6 (GRK6) in colorectal carcinoma (CRC). The aim of the present study was to investigate and examine the clinical value of GRK6 expression in human CRC. The expression of the GRK6 protein was determined in CRC tissues (n=83) and in normal colorectal tissues (n=19) by immunohistochemical (IHC) analysis. In addition, reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was conducted to investigate GRK6 mRNA levels in matched pairs of cancerous and non-cancerous fresh frozen tissues from 19 patients with CRC. Furthermore, GRK6 protein levels were evaluated in matched pairs of cancerous and non-cancerous fresh frozen tissues from 19 other patients with CRC by western blot analysis. The expression of GRK6 was significantly upregulated in patients with CRC as indicated by IHC analysis (P=0.028). The results of RT-qPCR and western blotting confirmed that GRK6 mRNA and protein levels were upregulated in CRC tissues compared with matched adjacent non-cancerous tissues (P<0.05). Additionally, GRK6 protein expression was significantly associated with histological differentiation (P=0.001), lymph node invasion (P=0.45), venous invasion (P=0.009), depth of invasion (P=0.026), distant metastasis (P<0.0001) and TNM stages (P=0.020). Survival analysis using the Kaplan-Meier method indicated that patients with high GRK6 expression levels exhibited lower overall survival rates compared with patients with low GRK6 expression. Multivariate analysis using the Cox proportional hazards model indicated that the expression levels of GRK6 (P=0.003) were independent prognostic factors for overall survival in patients. The overexpression of GRK6 in patients with CRC may serve as an independent predictor of patient outcome.

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.

Secreted miR-27a Induced by Cyclic Stretch Modulates the Proliferation of Endothelial Cells in Hypertension via GRK6

Abnormal proliferation of endothelial cells (ECs) is important in vascular remodeling during hypertension, but the mechanisms are still unclear. In hypertensive rats caused by abdominal aortic coarctation, the expression of G-protein-coupled receptor kinase 6 (GRK6) in ECs at common carotid artery was repressed in vivo, and EC proliferation was increased. 15% cyclic stretch in vitro, which mimics the pathologically increased stretch in hypertension, repressed EC GRK6 expression via paracrine control by vascular smooth muscle cells (VSMCs). Furthermore, VSMC-derived microparticles (VSMC-MPs) were detected in the conditioned medium from VSMCs and in artery. VSMC-MPs from cells exposed to 15% cyclic stretch decreased GRK6 expression and increased EC proliferation. miR-27a was detected in VSMC-MPs and was upregulated by 15% cyclic stretch. miR-27a was transferred from VSMCs to ECs via VSMC-MPs and directly targeted on GRK6. Finally, a multi-point injection of antagomiR-27a around carotid artery decreased miR-27a expression in vivo, induced GRK6 expression, and reversed the abnormal EC proliferation. Pathologically elevated cyclic stretch increased the secretion of miR-27a, which was transferred from VSMCs to ECs via the VSMC-MPs, subsequently targeted GRK6, and induced EC proliferation. Locally decreasing miR-27a could be a novel therapeutic approach to attenuate the abnormal EC proliferation in hypertension.

Effect of G Protein-coupled Receptor Kinase 6 on Proliferation and Apoptosis of Multiple Myeloma Cells and Its Mechanisms

To study the effect of G protein-coupled receptor kinase 6(GRK6) on proliferation and apoptosis of multiple myeloma(MM) cells and its mechanisms. The samples were collected from MM patients and healthy people for study in vivo. The plasma cells isolated from multiple myeloma patients, as well as U266 and NCI H929 myeloma cell lines were used for study in vitro. Western blot and quantitative real-time PCR were used to evaluate the protein and mRNA of expression of GRK6 in multiple myeloma, cell proliferation and apoptosis were tested by BrdU and Annexin V-FITC/PI assays. The protein and mRNA expression of GRK6 in multiple myeloma was higher than those in control group, and the expression level of GRK6 in stage I of MM was higher than that in control group, while the expression level of GRK6 in stage II was higher than that in stage I, but lower than that in stage III (P<0.05). U266 and MM cells showed high-sensitivity to CX-4945, except NCI H929. GRK6 expression level in U266, NCI H929 and MM cells treated with siRNA and CX-4945, significantly decreased as compared with those cells treated by CX-4945 alone. Cell proliferations of U266, NCI H92 and MM groups treated with CX-4945 were (58.25±18.24)%, (64.32±20.03)% and (45.42±25.01)% respectively, moreover, their apoptotic rates were (62.82±53.21)%, (43.25±47.05)% and (85.67±40.32)% respectively. The expression level of GRK6 in multiple myeloma increases, and GRK6 inhibitor CX-4945 inhibits proliferation and promotes apoptosis of myeloma cells; GRK6 regulates Rac1 and involves in the proliferation and apoptosis pathway of multiple myeloma cells.

SCF/c-kit transactivates CXCR4-serine 339 phosphorylation through G protein-coupled receptor kinase 6 and regulates cardiac stem cell migration.

C-kit positive cardiac stem cells (CSCs) have been shown to contribute to myocardial regeneration after infarction. Previously, we have shown that the c-kit ligand stem cell factor (SCF) can induce CSC migration into the infarcted area during myocardial infarction (MI). However, the precise mechanism involved is not fully understood. In this study, we found that CSCs also express C-X-C chemokine receptor type 4 (CXCR4), which is a typical member of the seven transmembrane-spanning G protein-coupled receptor (GPCR). In vitro, activation of c-kit signalling by SCF promotes migration of CSCs with increased phosphorylation of CXCR4-serine 339, p38 mitogen-activated protein kinase (p38 MAPK) and extracellular regulated protein kinases 1/2 (ERK1/2). Knockdown of CXCR4 expression by siRNA reduces SCF/c-kit-induced migration and downstream signalling. As previously reported, CXCR4-serine 339 phosphorylation is mainly regulated by GPCR kinase 6 (GRK6); thus, silencing of GRK6 expression by siRNA impairs CXCR4-serine 339 phosphorylation and migration of CSCs caused by SCF. In vivo, knockdown of GRK6 impairs the ability of CSCs to migrate into peri-infarcted areas. These results demonstrate that SCF-induced CSC migration is regulated by the transactivation of CXCR4-serine 339 phosphorylation, which is mediated by GRK6.

Effect of Tianqi antitremor granules on behavioral manifestations and expression of G protein-coupled receptor kinase 6 and &amp;beta;-arrestin1 in levodopa-induced dyskinesia in a rat model of Parkinson&amp;#39;s disease

BackgroundRecent studies have shown that expression of G protein-coupled receptor kinase 6 (GRK6) and β-arrestin1 in the striatum is closely associated with hyperactive dopamine receptors in rats with levodopa-induced dyskinesia (LID). Our research group has shown that Tianqi antitremor granules have a significant effect on the motor complications of Parkinson’s disease (PD). However, whether Tianqi antitremor granules have an effect on the behavioral manifestations and expression of GRK6 and β-arrestin1 in rats with LID is unknown.MethodsRats with PD received twice daily intraperitoneal injections of levodopa for 4 weeks to induce dyskinesia. Rats with LID were randomly divided into five groups: an LID-control group, an LID group, a levodopa plus Tianqi antitremor granules as traditional Chinese medicine (TCM)-low group, a levodopa plus TCM-medium group, and levodopa plus TCM-high group. Peak intensity of rotations was measured. GRK6 and β-arrestin1 expression in the striatum of the dyskinetic rats was observed by immunohistochemistry and Western blotting.ResultsPulsatile treatment with levodopa induced abnormal involuntary movements in rats with PD similar to LID in patients with PD. We found that repeated levodopa administration increased peak rotations in dyskinetic rats. However, peak rotations were decreased in rats given levodopa plus the different doses of Tianqi antitremor granules. In accordance with changed behavior, GRK6 and β-arrestin1 expression was decreased in rats with PD and was persistently low in rats with LID, but this decrease was prevented by coadministration of levodopa and Tianqi antitremor granules.ConclusionTianqi antitremor granules ameliorated levodopa-induced dyskinetic behavior, reversed the decrease in GRK6 and β-arrestin1 expression, and acted as a useful adjunctive medicine for the treatment of LID.

GRK6 expression in patients with hepatocellular carcinoma

ObjectiveTo investigate the expression and potential roles of G protein-coupled receptor kinase 6 (GRK6) in hepatocellular carcinoma (HCC) patients. MethodsImmunohistochemistry and Western blot was performed to determine GRK6 expression in 73 HCC samples. And the correlation with clinicopathological features was also analyzed. ResultsGRK6 expression was significantly higher in HCC than that in normal hepatic tissue. GRK6 was positively correlated with proliferation marker Ki-67, clinical stage, metastasis and survival time. ConclusionsOur results suggested that GRK6 overexpression plays an important role in HCC. Monitoring the expression of GRK6 maybe helpful in early diagnosis and prognosis of HCC.

Expression of G-protein-coupled receptor kinase 6 (GRK6) after acute spinal cord injury in adult rat

Spinal cord injury frequently results in permanent loss of neurological function. It includes many complex molecular and biochemical mechanisms. G-protein-coupled receptor kinase 6 (GRK6) is an intracellular kinase that regulates the sensitivity of certain G-protein-coupled receptors. Some studies reported GRK2 and GRK5 modulate the NFκB pathway in macrophages. Additionally, GRK2 is referred to as regulating activation of spinal cord microglia and GRK6 expression is significantly elevated in most brain regions in the MPTP-lesioned parkinsonian monkeys. However, the expression and function of GRK6 in nervous system lesion and repair are not well understood. In this study, we performed an acute spinal cord injury (SCI) model in adult rats. Western blot analysis showed the expression of GRK6 was upregulated significantly at protein level in spinal cord after SCI. Immunohistochemistry and immunofluorescence revealed wide expression of GRK6 in the normal spinal cord. After injury, GRK6 expression was increased predominantly in microglia, which expressed F4/80 (marker of macrophages and activated microglia) strongly. To understand whether GRK6 played a role in microglia activation, we applied lipopolysaccharide (LPS) to induce microglia activation in vitro. Western blot analysis demonstrated up-regulation in GRK6 protein expression after LPS stimulation was time- and dose-dependent and that up-regulation in F4/80 expression was concomitant with GRK6. These data suggested that GRK6 might be involved in the pathophysiology of SCI.

Effects of Chinese herbal medicine Tianqi Pingchan Granule on G protein-coupled receptor kinase 6 involved in the prevention of levodopa-induced dyskinesia in rats with Parkinson disease

To investigate the effects of Tianqi Pingchan (TQPC) Granule, a compound traditional Chinese herbal medicine with antitremor activity, on levodopa-induced dyskinesia and the expression of G protein-coupled receptor kinase 6 (GRK6) in rats with Parkinson disease (PD). The hemi-Parkinsonian rat model was established by sterotaxically injecting 6-hydroxydopa (6-OHDA) to the right medial forebrain bundle. Rats with PD were randomly divided into 5 groups with 5 in each. PD group was intraperitoneally injected with vitamin C; levodopa group was intraperitoneally injected with levodopa and benserazide; low-, medium- and high-dose TQPC Granule groups were intraperitoneally injected with levodopa and benserazide and treated with different dosages of TQPC Granule by gavage for 29 d. Another 5 rats were served as control with sham-operation. The behaviors of rats were observed and classified with abnormal involuntary movement (AIM) score. The expression of GRK6 in the striate of rats was detected by immunohistochemical method and Western blotting. AIM score was increased and the expression of GRK6 protein in lesion side was decreased after the long-tern treatment with levodopa and benserazide in rats. The AIM scores of rats with PD were decreased after TGPC Granule treatment. Immunohistochemical results showed that the number of GRK6-positive cells in medium- and high-dose TQPC Granule groups was increased as compared to that in the levodopa group (P<0.05). The expression level of GRK6 protein was increased in medium-dose TQPC Granule group when compared with the levodopa group (P<0.01), which was observed by Western blotting. TGPC Granule can increase the expression of GRK6, inhibit the increase of AIM, and reduce the incidence of levodopa-induced dyskinesia in rats with PD.

Endogenous G Protein-coupled Receptor Kinase 6 Regulates M3 Muscarinic Acetylcholine Receptor Phosphorylation and Desensitization in Human SH-SY5Y Neuroblastoma Cells

We have previously shown that overexpression of G protein-coupled receptor kinase 6 (GRK6) enhanced the phosphorylation and desensitization of the endogenously expressed M(3) muscarinic acetylcholine (mACh) receptor in human SH-SY5Y neuroblastoma cells. In this study we have examined the potential role of endogenous GRK6 in the regulation of M(3) mACh receptor by blocking its action through the introduction of a kinase-dead, dominant-negative GRK6 ((K215R)GRK6). (K215R)GRK6 expression inhibited methacholine-stimulated M(3) mACh receptor phosphorylation by 50% compared with plasmid transfected control cells. Guanosine-5'-O-(3-[(35)S]thio)triphosphate binding and immunoprecipitation studies, conducted after agonist pretreatment (3 min), indicated that M(3) mACh receptor-G alpha(q/11) uncoupling was attenuated by 50% in cells expressing (K215R)GRK6 when compared with control cells. In contrast, expression of the related dominant-negative kinase (K215R)GRK5 had no effect on M(3) mACh receptor phosphorylation or uncoupling. Time course studies also showed that agonist-stimulated [(3)H]inositol phosphate accumulations were more sustained in cells expressing (K215R)GRK6 compared with control and (K215R)GRK5-expressing cells, whereas (K215R)GRK6 expression had no effect on the phospholipase C response to direct stimulation of G proteins with AlF(4)(-). The ability of (K215R)GRK6 to inhibit agonist-mediated M(3) mACh receptor phosphorylation and G protein uncoupling suggests that endogenous GRK6 mediates, at least in part, M(3) mACh receptor desensitization in the SH-SY5Y cell line.