The Experts below are selected from a list of 498 Experts worldwide ranked by ideXlab platform
Robin A. Felder - One of the best experts on this subject based on the ideXlab platform.
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abstract 518 loss of caveolin 1 in mouse rat and human proximal tubule cells increases GRK4 activity and decreases d1 like receptor cell surface levels
Hypertension, 2014Co-Authors: John J Gildea, Dora Bigler Wang, Robert E Van Sciver, Robin A. FelderAbstract:Normal dopaminergic signaling in the renal proximal tubule under conditions of sodium loading leads to natriuresis without the need to increase blood pressure. Low caveolin-1 (CAV1) expression in the kidney is associated with dopaminergic signaling defects and salt sensitive hypertension in both mice and rats. We and others have shown that SNPs in human CAV1 are associated with hypertension. We hypothesize that low CAV1 expression causes increased GRK4 kinase activity due to lack of physical interaction and inhibition of GRK4; thus overactive GRK4 would inactivate the dopamine-1 receptor (D 1 R). We have previously shown that CAV1 reduction, knockdown or knockout in mouse, rat and human renal proximal tubule cells (RPTC) leads to decreased dopamine D 1 -like receptor binding at the proximal tubule cell surface and that re-expression of CAV1 alpha isoform is necessary to rescue this dopaminergic defect. Here we extend those studies by showing that defects in D 1 R cell surface expression due to low CAV1 expression can also be rescued by inhibiting GRK4 in human RPTC using GRK4 siRNA or by introducing heparin (1 μmol/L, 24 hrs) in mouse and rat RPTC. GRK4 siRNA lowered GRK4 expression by greater than 80% in all four human RPTC cell lines and partially restored fluorescent D 1 -like receptor antagonist bodipy630 SKF83566 cell surface binding (scrambled siRNA 66,231±7,286 RFU, GRK4 siRNA 96,206±11,035 RFU, P 1 -like receptor surface expression. Electroporation of heparin in these CAV1 transfected cells (SHR CAV1) restored bodipy630 SKF83566 cell surface binding back to levels found in WKY RPTC (SHR CAV1 35,241±4,280 RFU, SHR CAV1 heparin 54,218±6,564 RFU, WKY 56,821± 3,546 RFU, P 1 -like receptor defects caused by loss of CAV1 are likely due to increased GRK4 activity and can be restored by either increasing CAV1 expression or decreasing GRK4 expression.
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abstract 517 ubiquitylation of g protein coupled receptor kinase 4 GRK4 regulates its localization and abundance
Hypertension, 2014Co-Authors: John J Gildea, Dora Bigler Wang, Walid Omer, Hanh T Tran, Robin A. FelderAbstract:The dopamine-1 receptor (D 1 R) is responsible for regulating up to 60% of natriuresis in the kidney under sodium loaded conditions. G protein coupled receptor kinase type 4 (GRK4) phosphorylates the D 1 R and reduces its membrane expression, but little is known about regulation of GRK4. We hypothesized that GRK4 is targeted for elimination through ubiquitylation. Using D 1 R and GRK4γ stably transfected human embryonic kidney cells (HEK293), we demonstrated GRK4 and ubiquitin coimmunoprecipitation. Addition of clasto-lactacystin beta-lactone (CLBL, an inhibitor of the 26S proteasome) increased GRK4 expression levels (western blot) using two different GRK4 fusion protein constructs. The addition of CLBL (10 μmol/L, 24 hrs) increased the expression of both a tandem affinity tagged (tap-tag) GRK4 fusion construct (2.65±0.19 fold over vehicle (VEH): VEH 4,010±404 RFU; CLBL 10,626±744 RFU, P Ub K Ub LQKK) are located near the nuclear localization signal (AA 219-228; QKKRIKKRK) in GRK4. We therefore examined the subcellular localization of GRK4 after CLBL treatment. Nuclear accumulation of the GRK4-mCherry fusion protein fluorescence decreased markedly while cytoplasmic GRK4-mCherry fluorescence increased (nuclear to cytoplasmic GRK4 ratio: VEH 1.18±0.09 RFU; CLBL 0.78±0.03 RFU, P
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abstract 532 GRK4 palmitoylation is necessary for membrane association and dopamine 1 receptor activity
Hypertension, 2014Co-Authors: John J Gildea, Dora Bigler Wang, Hanh T Tran, Zhenyue Huang, Robin A. FelderAbstract:The renal dopaminergic system regulates a large percentage of renal sodium excretion under sodium loaded conditions; however, not much is known about intracellular dopaminergic regulatory mechanisms. The expression of the principal sodium regulatory dopaminergic receptor (the D1R) is negatively modulated by the G protein linked receptor kinase type 4 (GRK4) through phosphorylation while the D1R is expressed in the plasma membrane. Therefore, we hypothesized that mechanisms associated with GRK4 membrane localization might be associated with its activity. Using human renal proximal tubule cell lines (RPTC), we previously demonstrated that GRK4 is palmitoylated using 17-octadecynoic acid (17-ODYA) and click chemistry and that fenoldopam (FEN, D1R agonist) decreased palmitoylation only in normal cAMP-coupled RPTC. Here we expand on those studies by showing that GRK4 palmitoylation was inhibited by 2-bromopalmitate (2BP, 100 μmol/L, palmitoylation inhibitor) which shifted GRK4 expression from the membrane to the cytoplasm. We used two different techniques to show loss of GRK4 membrane association when GRK4 palmitoylation was inhibited. The first technique involved classic cell fractionation using ultracentrifugation and western blot identification of GRK4 in membrane protein fractions (VEH 0.174±0.023 RFU, 2BP 0.091±0.013 RFU, P
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Abstract 467: Hypertension and Cancer: Are They Related?
Hypertension, 2013Co-Authors: Hanh T Tran, John J Gildea, Robin A. FelderAbstract:There are literature references supporting a link between hypertension and cancer. However, a mechanism for this association has not been defined. Key players in blood pressure regulation include the dopamine-1 receptor (D 1 R), G protein-coupled kinase type 4 (GRK4) and c-Myc, all found in renal proximal tubule cells (RPTC). c-Myc also regulates GRK4 in the breast cancer cell line MCF7. We hypothesized that the D 1 R would act as a tumor suppressor and both GRK4 and c-Myc as oncogenes in MCF7 cells. GRK4 is positively regulated transcriptionally by c-Myc, and GRK4 is negatively regulated by Caveolin-1 (CAV1) in RPTCs, so we tested if the same relationship occurs in this breast cancer model. SKF38393 (D 1 R agonist, 10μM, 18hr) reduced wild type MCF7 (WT-MCF7) cell motility (an index of aggressive growth) by 16.0% ± 3.2 SEM (n=9; p 1 R antagonist LE300 (10μM, 18hr) increased cell motility 23.0% ± 7.0 SEM (n=9; p 1 R (103.3% ± 35.9 SEM; n=10; p 1 R membrane recruitment post-fenoldopam stimulation (FEN, D 1 R agonist, 10μM, 30min) (increase of 65.5% ± 6.18 SEM; n=4; p
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Abstract 79: Caveolin-1 Knockout Mice Have Salt-Sensitive Hypertension and Dopamine-1 Receptor Defects in Renal Cortex and Isolated Renal Proximal Tubule Cells
Hypertension, 2013Co-Authors: John J Gildea, Robert E Van Sciver, Nancy L. Howell, Brandon A. Kemp, Robert M. Carey, Robin A. FelderAbstract:Dopamine-1 receptors (D1R) are necessary for kidney proximal tubule-dependent natriuresis and maintenance of normal blood pressure, especially under high salt conditions. G-protein coupled receptor kinase 4 (GRK4) is a negative regulator of D1R function and single nucleotide polymorphisms in GRK4 have been associated with both hypertension and salt sensitivity in humans. Caveolin-1 (CAV1) directly binds to GRK4 and decreases kinase activity. We hypothesized that CAV1 knockout mice (CAV1KO) would have increased GRK4 kinase activity due to lack of physical interaction and inhibition of GRK4; thus overactive GRK4 would inactivate the D1R. Mean arterial blood pressure (MAP, in mmHg ±SEM) measured over 5 days was not significantly different for Wild-type mice (WT, 128.9±4.2 mmHg, n=4) vs CAV1KO (129.5±3.5 mmHg, n=4) on normal chow (0.3% sodium). However, on a 4% high sodium diet, the MAP of CAV1KO mice increased in just 2 days by 20.1±4.2 mmHg (p
Pedro A. Jose - One of the best experts on this subject based on the ideXlab platform.
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THE ROLE OF G PROTEIN-COUPLED RECEPTOR KINASE 4 IN CARDIOMYOCYTE INJURY AFTER MYOCARDIAL INFARCTION
Journal of Hypertension, 2019Co-Authors: L. Li, Pedro A. Jose, L. Tang, S. Qu, D. Yang, Q. Liao, W. Fu, W. Wang, C ZengAbstract:Objective:G-protein-coupled receptor kinase 4 (GRK4) has been reported to play a regulatory role in hypertension, but little is known of its role in cardiomyocytes and myocardial infarction (MI). The goal of present study is to explore the role of cardiac-restricted GRK4 overexpression in the pathog
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abstract p187 the salt sensitivity of human GRK4 transgenic mice is associated with renal oxidative stress
Hypertension, 2015Co-Authors: Zhenyu Diao, Laureano D. Asico, Pedro A. Jose, Van Anthony M Villar, Xiaoxu Zheng, Santiago Cuevas, Ines Armando, Xiaoyan WangAbstract:Variants of hGRK4 gamma are associated with human essential hypertension. hGRK4gamma 142V transgenic mice are hypertensive without oxidative stress on normal salt intake. hGRK4 gamma 486V transgenic mice have salt-sensitive hypertension. Because renal oxidative stress is increased in some rodent models of salt-sensitive hypertension, we quantified the renal expression of reactive oxygen species related proteins in hGRK4 gamma 486V transgenic mice and non-transgenic (NT) littermates, on normal (NS, 0 . 8% ) and high (HS, 4%) NaCl diets . Systolic blood pressure (measured under anesthesia) was similar in hGRK4 gamma 486V (89.83±2.7, mm Hg, n=9) and NT (94.7±2.5) mice on NS diet and elevated in hGRK4 gamma 486V (114±6.1) but not in NT mice (94.1±2.8) on HS diet. The renal expressions of NOX1, 2, and 4 were similar in both strains on NS diet but NOX2 was decreased by HS in NT (28±7, % of NT on NS diet, n=5-6/group). On NS diet, CuZnSOD and ECSOD were similar in the two mouse strains while MnSOD (66±3%) was lower in GRK4 gamma 486V than NT mice. However, on HS diet, CuZnSOD (87±7%), MnSOD (70±3%), and ECSOD (55±7%) were decreased in GRK4 gamma 486V mice but not altered in NT mice. HO-2, not HO-1, was slightly greater in GRK4 gamma 486V than NT mice on NS diet (117±7%) but this difference was abolished by HS diet. Urinary 8-isoprostane was lower in GRK4 gamma 486V than NT mice (57±2.5 vs 70±0.1, ng/mg of Cr) on NS diet but increased to a greater extent in GRK4 gamma 486V than NT mice (197±18 vs128±6) on HS diet. Renal SOD activity and superoxide production were similar in both mouse strains on NS diet. HS diet decreased SOD activity (81.6±2.7%) and increased superoxide (138.9±6.6%) production in GRK4 gamma 486V mice but not in NT mice. The renal tubular immunofluorescence of ECSOD was reduced to a greater extent in hGRK4 gamma 486V than NT by HS diet. HS diet also decreased the renal expression of NOS3, not NOS 1 and 2, in GRK4 gamma 486V mice (49±2%) but not in NT mice. These changes were not observed in the mice with hGRK4 gamma wildtype and hGRK4 gamma 142V transgenic mice, suggesting that the salt-sensitive hypertension of GRK4 gamma 486V mice is related to renal oxidative stress.
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Abstract 303: Decreased Adrenomedullin and Interaction with NHE3 in Human GRK4γ142v Transgenic Mice
Hypertension, 2014Co-Authors: Donghai Zhou, John E. Jones, Laureano D. Asico, Pedro A. Jose, Xiaoyan WangAbstract:The expression of the human G protein-coupled receptor kinase 4γ (GRK4γ) 142V variant (hGRK4γ 142V ) in mice causes hypertension that is related to dysfunction of renal dopaminergic and renin-angiotensin systems. To determine if there are other dysfunctional systems in hGRK4γ 142V mice, we quantified the expression of adrenomedullin (ADM), a natriuretic hormone, and components of its receptor, RAMP2 (receptor activity-modified protein2), CLCR (calcitonin-like calcitonin receptor), and ADMRL2 (ADM receptor L2) in the kidneys of hGRK4 γ 142V and hGRK4 γ WT transgenic mice and non-transgenic (NT) littermates . Systolic blood pressure (measured under anesthesia) was elevated in hGRK4 γ 142V (129.3±10.4, mm Hg) but not in GRK4 γ WT mice (101.7±6.3), relative to NT littermates (99.8±1.8) (n=4/group). The sodium excretion and blood pressure plot in GRK4 γ 142V mice was shifted to the right of GRK4 γ WT mice. Protein expressions of ADM (37±4% of NT by immunoblotting and immunofluorescence) and RAMP2 (36±16%) were decreased in hGRK4 γ 142V mice but unchanged in GRK4 γ WT mice, relative to NT littermates. The protein expressions of CLCR and ADMRL2 were not altered in hGRK4 γ 142V and GRK4 γ WT . In the kidney ADM colocalized with NHE3 but not with NKCC2 and NCC. NHE3 protein expression in renal homogenates and brush border membranes was not altered in GRK4 γ 142V mice. However, the amount of NHE3 that coimmunoprecipitated with ADM was decreased in GRK4 γ 142V (77±3% of NT) mice; a similar decrease was also found in the reverse-coimmunoprecipitation of NHE3 with ADM. In vitro , human ADM inhibited apical sodium transport in polarized human renal proximal tubule cells (1 hr, n=5-6) at concentrations of 10 nM (84±4 % of vehicle) and 100 nM (81±3%), similar to that achieved with the NHE3 inhibitor EIPA (81±6%, 100 nM), suggesting that NHE3 activity was inhibited by ADM. Our data suggest that GRK4γ 142V decreases ADM/RAMP2 expression in the kidney; the natriuretic effect of ADM may depend mainly on the inhibition of the activities of sodium transporters along the nephron (e.g., NHE3). The decreased ADM/NHE3 interaction and dysfunctional natriuretic and antinatriuretic factors may contribute to the impaired sodium excretion and increased blood pressure in GRK4 γ 142V transgenic mice.
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abstract 271 role of arterial at1 receptor on the regulation of GRK4 in hypertension
Circulation Research, 2014Co-Authors: Ken Chen, Pedro A. Jose, Caiyu Chen, Chunyu ZengAbstract:G protein-coupled receptor kinase 4 (GRK4) gene variants or increased GRK4 expression, via impairment of renal dopamine receptor and enhancement of renin angiotensin system function, impairs renal sodium excretion, resulting in sodium retention and an increase in blood pressure. Increased aortic stiffness, a risk factor in cardiovascular disease, may be related to increased activity of the renin-angiotensin system Whether or not GRK4 and the angiotensin type 1 receptor (AT1R) interact in the aorta is not known. We now report that GRK4 is expressed in vascular smooth muscle cells (VSMCs) of the aorta. Exogenous expression of the GRK4 variant 142V in aortic A10 cells increased AT1R protein expression and AT1R-mediated increase in intracellular calcium concentration. The increased AT1R expression was caused by an increase in AT1R mRNA expression via NF-kappa B, because blockade of NF-kappa B abolished those effects of GRK4 A142V. As compared with control (vector-transfected) cells, cells expressing 142V had higher NF-kappa B activity and more NF-kappa B bound to AT1R promoter. The increased AT1R expression in cells expressing GRK4 142V was associated with decreased AT1R degradation, which was ascribed to the lower AT1R phosphorylation. There was direct interaction between GRK4 and AT1R in A10 cells which was decreased by GRK4 that could have caused the lower AT1R phosphorylation and degradation. The regulation of GRK4 of AT1R expression was confirmed in GRK4142V transgenic mice, the AT1R expression was higher, while AT1R phosphorylation was lower in aorta in GRK4 142V than control mice. Angiotensin II-mediated vasoconstriction was higher in A142V mice. This study provides a mechanism that GRK4, via regulation of arterial AT1R expression and function, engaged into the pathogenesis of hypertension.
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role of GRK4 in the regulation of arterial at1 receptor in hypertension
Hypertension, 2014Co-Authors: Ken Chen, Pedro A. Jose, Chunjiang Fu, Caiyu Chen, Jian Yang, Duofen He, Lin Zhou, Zhiwei Yang, Lianfeng Zhang, Chunyu ZengAbstract:G-protein–coupled receptor kinase 4 (GRK4) gene variants, via impairment of renal dopamine receptor and enhancement of renin–angiotensin system functions, cause sodium retention and increase blood pressure. Whether GRK4 and the angiotensin type 1 receptor (AT 1 R) interact in the aorta is not known. We report that GRK4 is expressed in vascular smooth muscle cells of the aorta. Heterologous expression of the GRK4γ variant 142V in A10 cells increased AT 1 R protein expression and AT 1 R-mediated increase in intracellular calcium concentration. The increase in AT 1 R expression was related to an increase in AT 1 R mRNA expression via the NF-κB pathway. As compared with control, cells expressing GRK4γ 142V had greater NF-κB activity with more NF-κB bound to the AT 1 R promoter. The increased AT 1 R expression in cells expressing GRK4γ 142V was also associated with decreased AT 1 R degradation, which may be ascribed to lower AT 1 R phosphorylation. There was a direct interaction between GRK4γ and AT 1 R that was decreased by GRK4γ 142V. The regulation of AT 1 R expression by GRK4γ 142V in A10 cells was confirmed in GRK4γ 142V transgenic mice; AT 1 R expression was higher in the aorta of GRK4γ 142V transgenic mice than control GRK4γ wild-type mice. Angiotensin II–mediated vasoconstriction of the aorta was also higher in GRK4γ 142V than in wild-type transgenic mice. This study provides a mechanism by which GRK4, via regulation of arterial AT 1 R expression and function, participates in the pathogenesis of conduit vessel abnormalities in hypertension.
Richard T Premont - One of the best experts on this subject based on the ideXlab platform.
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Once and future signaling
NeuroMolecular Medicine, 2005Co-Authors: Richard T PremontAbstract:G protein-coupled receptors (GPCRs) are the most numerous class of cell surface receptor, and substances acting through GPCRs mediate many critical signaling events and physiological processes. GPCR sensitivity and signaling is dynamic, responding rapidly to adjust to changes in the ambient level of stimulation of target cells. One important mediator of such receptor sensitivity is the family of GPCR kinases (GRKs). Like heterotrimeric G proteins, GRKs recognize agonist-bound, activated receptors, and this recognition promotes catalytic activation of GRKs, resulting in the preferential phosphorylation of activated receptors. GRK-phosphorylated receptors are then targeted by arrestin proteins, which bind to phosphorylated receptors. Arrestin-bound receptors are uncoupled from heterotrimeric G proteins, resulting in decreased sensitivity to further receptor stimulation (desensitization). Arrestin-bound receptors are also accelerated into internalization pathways and linked to distinct arrestin-mediated signaling pathways. GRKs thus serve as gatekeepers for receptors, terminating some signaling pathways and initiating others. One major outstanding question concerning GRKs understanding the mechanisms by which any particular receptor subtype (of the 800 or so in the body) is regulated by a specific GRK(s), and the consequences of this specificity. An understanding of this regulatory specificity could allow targeting of GRK function to ameliorate diseases involving GPCR dysregulation.
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the GRK4 subfamily of g protein coupled receptor kinases alternative splicing gene organization and sequence conservation
Journal of Biological Chemistry, 1999Co-Authors: Richard T Premont, Alexander D Macrae, Samuel A J R Aparicio, Humphrey E Kendall, Jeffrey E Welch, Robert J LefkowitzAbstract:Abstract G protein-coupled receptor kinases (GRKs) desensitize G protein-coupled receptors by phosphorylating activated receptors. The six known GRKs have been classified into three subfamilies based on sequence and functional similarities. Examination of the mouse GRK4 subfamily (GRKs 4, 5, and 6) suggests that mouse GRK4 is not alternatively spliced in a manner analogous to human or rat GRK4, whereas GRK6 undergoes extensive alternative splicing to generate three variants with distinct carboxyl termini. Characterization of the mouse GRK 5 and 6 genes reveals that all members of the GRK4 subfamily share an identical gene structure, in which 15 introns interrupt the coding sequence at equivalent positions in all three genes. Surprisingly, none of the three GRK subgroups (GRK1, GRK2/3, and GRK4/5/6) shares even a single intron in common, indicating that these three subfamilies are distinct gene lineages that have been maintained since their divergence over 1 billion years ago. Comparison of the amino acid sequences of GRKs from various mammalian species indicates that GRK2, GRK5, and GRK6 exhibit a remarkably high degree of sequence conservation, whereas GRK1 and particularly GRK4 have accumulated amino acid changes at extremely rapid rates over the past 100 million years. The divergence of individual GRKs at vastly different rates reveals that strikingly different evolutionary pressures apply to the function of the individual GRKs.
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Phosphatidylinositol 4,5-Bisphosphate (PIP2)-enhanced G Protein-coupled Receptor Kinase (GRK) Activity: LOCATION, STRUCTURE, AND REGULATION OF THE PIP2 BINDING SITE DISTINGUISHES THE GRK SUBFAMILIES
Journal of Biological Chemistry, 1996Co-Authors: J A Pitcher, Walter J. Koch, Richard T Premont, Zl Fredericks, W C Stone, Robert H. Stoffel, Robert J LefkowitzAbstract:Abstract The G protein-coupled receptor kinases (GRKs) phosphorylate agonist occupied G protein-coupled receptors and play an important role in mediating receptor desensitization. The localization of these enzymes to their membrane incorporated substrates is required for their efficient function and appears to be a highly regulated process. In this study we demonstrate that phosphatidylinositol 4,5-bisphosphate (PIP2) enhances GRK5-mediated β-adrenergic receptor (βAR) phosphorylation by directly interacting with this enzyme and facilitating its membrane association. GRK5-mediated phosphorylation of a soluble peptide substrate is unaffected by PIP2, suggesting that the PIP2-enhanced receptor kinase activity arises as a consequence of this membrane localization. The lipid binding site of GRK5 exhibits a high degree of specificity and appears to reside in the amino terminus of this enzyme. Mutation of six basic residues at positions 22, 23, 24, 26, 28, and 29 of GRK5 ablates the ability of this kinase to bind PIP2. This region of the GRK5, which has a similar distribution of basic amino acids to the PIP2 binding site of gelsolin, is highly conserved between members of the GRK4 subfamily (GRK4, GRK5, and GRK6). Indeed, all the members of the GRK4 subfamily exhibit PIP2-dependent receptor kinase activity. We have shown previously that the membrane association of βARK (β-adrenergic receptor kinase) (GRK2) is mediated, in vitro, by the simultaneous binding of PIP2 and the βγ subunits of heterotrimeric G proteins to the carboxyl-terminal pleckstrin homology domain of this enzyme (Pitcher, J. A., Touhara, K., Payne, E. S., and Lefkowitz, R. J. (1995) J. Biol. Chem.270, 11707-11710). Thus, five members of the GRK family bind PIP2, βARK (GRK2), βARK2 (GRK3), GRK4, GRK5, and GRK6. However, the structure, location, and regulation of the PIP2 binding site distinguishes the βARK (GRK2 and GRK3) and GRK4 (GRK4, GRK5, and GRK6) subfamilies.
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Members of the G protein-coupled receptor kinase family that phosphorylate the beta2-adrenergic receptor facilitate sequestration.
Biochemistry, 1996Co-Authors: Luc Ménard, Stephen S. G. Ferguson, Richard T Premont, Robert J Lefkowitz, Larry S. Barak, Lucie Bertrand, Anne-marie Colapietro, Marc G CaronAbstract:We recently reported that a β2-adrenergic receptor (β2AR) mutant, Y326A, defective in its ability to sequester in response to agonist stimulation was a poor substrate for G protein-coupled receptor kinase (GRK)-mediated phosphorylation; however, its ability to be phosphorylated and sequestered could be restored by overexpressing GRK2 [Ferguson et al. (1995) J. Biol. Chem. 270, 24782]. In the present report, we tested the ability of each of the known GRKs (GRK1−6) to phosphorylate and rescue the sequestration of the Y326A mutant in HEK-293 cells. We demonstrate that in addition to GRK2, GRK3−6 can phosphorylate the Y326A mutant and rescue its sequestration; however, GRK1 was totally ineffective in rescuing either the phosphorylation or the sequestration of the mutant receptor. We found that the agonist-dependent rescue of Y326A mutant phosphorylation by GRK2, -3, and -5 was associated with the agonist-dependent rescue of sequestration. In contrast, overexpression of GRK4 and -6 led mainly to agonist-indepe...
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characterization of the g protein coupled receptor kinase GRK4 identification of four splice variants
Journal of Biological Chemistry, 1996Co-Authors: Richard T Premont, Alexander D Macrae, Robert H. Stoffel, Julie A. Pitcher, Namjin Chung, Christine Ambrose, James Inglese, Marcy E Macdonald, Robert J LefkowitzAbstract:A novel human G protein-coupled receptor kinase was recently identified by positional cloning in the search for the Huntington's disease locus (Ambrose, C., James, M., Barnes, G., Lin, C., Bates, G., Altherr, M., Duyao, M., Groot, N., Church, D., Wasmuth, J. J., Lehrach, H., Housman, D., Buckler, A., Gusella, J. F., and MacDonald, M. E. (1993) Hum. Mol. Genet. 1, 697-703). Comparison of the deduced amino acid sequence of GRK4 with those of the closely related GRK5 and GRK6 suggested the apparent loss of 32 codons in the amino-terminal domain and 46 codons in the carboxyl-terminal domain of GRK4. These two regions undergo alternative splicing in the GRK4 mRNA, resulting from the presence or absence of exons filling one or both of these apparent gaps. Each inserted sequence maintains the open reading frame, and the deduced amino acid sequences are similar to corresponding regions of GRK5 and GRK6. Thus, the GRK4 mRNA and the GRK4 protein can exist as four distinct variant forms. The human GRK4 gene is composed of 16 exons extending over 75 kilobase pairs of DNA. The two alternatively spliced exons correspond to exons II and XV. The genomic organization of the GRK4 gene is completely distinct from that of the human GRK2 gene, highlighting the evolutionary distance since the divergence of these two genes. Human GRK4 mRNA is expressed highly only in testis, and both alternative exons are abundant in testis mRNA. The four GRK4 proteins have been expressed, and all incorporate [3H]palmitate. GRK4 is capable of augmenting the desensitization of the rat luteinizing hormone/chorionic gonadotropin receptor upon coexpression in HEK293 cells and of phosphorylating the agonist-occupied, purified beta2-adrenergic receptor, indicating that GRK4 is a functional protein kinase.
John J Gildea - One of the best experts on this subject based on the ideXlab platform.
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abstract 517 ubiquitylation of g protein coupled receptor kinase 4 GRK4 regulates its localization and abundance
Hypertension, 2014Co-Authors: John J Gildea, Dora Bigler Wang, Walid Omer, Hanh T Tran, Robin A. FelderAbstract:The dopamine-1 receptor (D 1 R) is responsible for regulating up to 60% of natriuresis in the kidney under sodium loaded conditions. G protein coupled receptor kinase type 4 (GRK4) phosphorylates the D 1 R and reduces its membrane expression, but little is known about regulation of GRK4. We hypothesized that GRK4 is targeted for elimination through ubiquitylation. Using D 1 R and GRK4γ stably transfected human embryonic kidney cells (HEK293), we demonstrated GRK4 and ubiquitin coimmunoprecipitation. Addition of clasto-lactacystin beta-lactone (CLBL, an inhibitor of the 26S proteasome) increased GRK4 expression levels (western blot) using two different GRK4 fusion protein constructs. The addition of CLBL (10 μmol/L, 24 hrs) increased the expression of both a tandem affinity tagged (tap-tag) GRK4 fusion construct (2.65±0.19 fold over vehicle (VEH): VEH 4,010±404 RFU; CLBL 10,626±744 RFU, P Ub K Ub LQKK) are located near the nuclear localization signal (AA 219-228; QKKRIKKRK) in GRK4. We therefore examined the subcellular localization of GRK4 after CLBL treatment. Nuclear accumulation of the GRK4-mCherry fusion protein fluorescence decreased markedly while cytoplasmic GRK4-mCherry fluorescence increased (nuclear to cytoplasmic GRK4 ratio: VEH 1.18±0.09 RFU; CLBL 0.78±0.03 RFU, P
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abstract 532 GRK4 palmitoylation is necessary for membrane association and dopamine 1 receptor activity
Hypertension, 2014Co-Authors: John J Gildea, Dora Bigler Wang, Hanh T Tran, Zhenyue Huang, Robin A. FelderAbstract:The renal dopaminergic system regulates a large percentage of renal sodium excretion under sodium loaded conditions; however, not much is known about intracellular dopaminergic regulatory mechanisms. The expression of the principal sodium regulatory dopaminergic receptor (the D1R) is negatively modulated by the G protein linked receptor kinase type 4 (GRK4) through phosphorylation while the D1R is expressed in the plasma membrane. Therefore, we hypothesized that mechanisms associated with GRK4 membrane localization might be associated with its activity. Using human renal proximal tubule cell lines (RPTC), we previously demonstrated that GRK4 is palmitoylated using 17-octadecynoic acid (17-ODYA) and click chemistry and that fenoldopam (FEN, D1R agonist) decreased palmitoylation only in normal cAMP-coupled RPTC. Here we expand on those studies by showing that GRK4 palmitoylation was inhibited by 2-bromopalmitate (2BP, 100 μmol/L, palmitoylation inhibitor) which shifted GRK4 expression from the membrane to the cytoplasm. We used two different techniques to show loss of GRK4 membrane association when GRK4 palmitoylation was inhibited. The first technique involved classic cell fractionation using ultracentrifugation and western blot identification of GRK4 in membrane protein fractions (VEH 0.174±0.023 RFU, 2BP 0.091±0.013 RFU, P
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abstract 518 loss of caveolin 1 in mouse rat and human proximal tubule cells increases GRK4 activity and decreases d1 like receptor cell surface levels
Hypertension, 2014Co-Authors: John J Gildea, Dora Bigler Wang, Robert E Van Sciver, Robin A. FelderAbstract:Normal dopaminergic signaling in the renal proximal tubule under conditions of sodium loading leads to natriuresis without the need to increase blood pressure. Low caveolin-1 (CAV1) expression in the kidney is associated with dopaminergic signaling defects and salt sensitive hypertension in both mice and rats. We and others have shown that SNPs in human CAV1 are associated with hypertension. We hypothesize that low CAV1 expression causes increased GRK4 kinase activity due to lack of physical interaction and inhibition of GRK4; thus overactive GRK4 would inactivate the dopamine-1 receptor (D 1 R). We have previously shown that CAV1 reduction, knockdown or knockout in mouse, rat and human renal proximal tubule cells (RPTC) leads to decreased dopamine D 1 -like receptor binding at the proximal tubule cell surface and that re-expression of CAV1 alpha isoform is necessary to rescue this dopaminergic defect. Here we extend those studies by showing that defects in D 1 R cell surface expression due to low CAV1 expression can also be rescued by inhibiting GRK4 in human RPTC using GRK4 siRNA or by introducing heparin (1 μmol/L, 24 hrs) in mouse and rat RPTC. GRK4 siRNA lowered GRK4 expression by greater than 80% in all four human RPTC cell lines and partially restored fluorescent D 1 -like receptor antagonist bodipy630 SKF83566 cell surface binding (scrambled siRNA 66,231±7,286 RFU, GRK4 siRNA 96,206±11,035 RFU, P 1 -like receptor surface expression. Electroporation of heparin in these CAV1 transfected cells (SHR CAV1) restored bodipy630 SKF83566 cell surface binding back to levels found in WKY RPTC (SHR CAV1 35,241±4,280 RFU, SHR CAV1 heparin 54,218±6,564 RFU, WKY 56,821± 3,546 RFU, P 1 -like receptor defects caused by loss of CAV1 are likely due to increased GRK4 activity and can be restored by either increasing CAV1 expression or decreasing GRK4 expression.
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Abstract 79: Caveolin-1 Knockout Mice Have Salt-Sensitive Hypertension and Dopamine-1 Receptor Defects in Renal Cortex and Isolated Renal Proximal Tubule Cells
Hypertension, 2013Co-Authors: John J Gildea, Robert E Van Sciver, Nancy L. Howell, Brandon A. Kemp, Robert M. Carey, Robin A. FelderAbstract:Dopamine-1 receptors (D1R) are necessary for kidney proximal tubule-dependent natriuresis and maintenance of normal blood pressure, especially under high salt conditions. G-protein coupled receptor kinase 4 (GRK4) is a negative regulator of D1R function and single nucleotide polymorphisms in GRK4 have been associated with both hypertension and salt sensitivity in humans. Caveolin-1 (CAV1) directly binds to GRK4 and decreases kinase activity. We hypothesized that CAV1 knockout mice (CAV1KO) would have increased GRK4 kinase activity due to lack of physical interaction and inhibition of GRK4; thus overactive GRK4 would inactivate the D1R. Mean arterial blood pressure (MAP, in mmHg ±SEM) measured over 5 days was not significantly different for Wild-type mice (WT, 128.9±4.2 mmHg, n=4) vs CAV1KO (129.5±3.5 mmHg, n=4) on normal chow (0.3% sodium). However, on a 4% high sodium diet, the MAP of CAV1KO mice increased in just 2 days by 20.1±4.2 mmHg (p
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Abstract 467: Hypertension and Cancer: Are They Related?
Hypertension, 2013Co-Authors: Hanh T Tran, John J Gildea, Robin A. FelderAbstract:There are literature references supporting a link between hypertension and cancer. However, a mechanism for this association has not been defined. Key players in blood pressure regulation include the dopamine-1 receptor (D 1 R), G protein-coupled kinase type 4 (GRK4) and c-Myc, all found in renal proximal tubule cells (RPTC). c-Myc also regulates GRK4 in the breast cancer cell line MCF7. We hypothesized that the D 1 R would act as a tumor suppressor and both GRK4 and c-Myc as oncogenes in MCF7 cells. GRK4 is positively regulated transcriptionally by c-Myc, and GRK4 is negatively regulated by Caveolin-1 (CAV1) in RPTCs, so we tested if the same relationship occurs in this breast cancer model. SKF38393 (D 1 R agonist, 10μM, 18hr) reduced wild type MCF7 (WT-MCF7) cell motility (an index of aggressive growth) by 16.0% ± 3.2 SEM (n=9; p 1 R antagonist LE300 (10μM, 18hr) increased cell motility 23.0% ± 7.0 SEM (n=9; p 1 R (103.3% ± 35.9 SEM; n=10; p 1 R membrane recruitment post-fenoldopam stimulation (FEN, D 1 R agonist, 10μM, 30min) (increase of 65.5% ± 6.18 SEM; n=4; p
Robert J Lefkowitz - One of the best experts on this subject based on the ideXlab platform.
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the GRK4 subfamily of g protein coupled receptor kinases alternative splicing gene organization and sequence conservation
Journal of Biological Chemistry, 1999Co-Authors: Richard T Premont, Alexander D Macrae, Samuel A J R Aparicio, Humphrey E Kendall, Jeffrey E Welch, Robert J LefkowitzAbstract:Abstract G protein-coupled receptor kinases (GRKs) desensitize G protein-coupled receptors by phosphorylating activated receptors. The six known GRKs have been classified into three subfamilies based on sequence and functional similarities. Examination of the mouse GRK4 subfamily (GRKs 4, 5, and 6) suggests that mouse GRK4 is not alternatively spliced in a manner analogous to human or rat GRK4, whereas GRK6 undergoes extensive alternative splicing to generate three variants with distinct carboxyl termini. Characterization of the mouse GRK 5 and 6 genes reveals that all members of the GRK4 subfamily share an identical gene structure, in which 15 introns interrupt the coding sequence at equivalent positions in all three genes. Surprisingly, none of the three GRK subgroups (GRK1, GRK2/3, and GRK4/5/6) shares even a single intron in common, indicating that these three subfamilies are distinct gene lineages that have been maintained since their divergence over 1 billion years ago. Comparison of the amino acid sequences of GRKs from various mammalian species indicates that GRK2, GRK5, and GRK6 exhibit a remarkably high degree of sequence conservation, whereas GRK1 and particularly GRK4 have accumulated amino acid changes at extremely rapid rates over the past 100 million years. The divergence of individual GRKs at vastly different rates reveals that strikingly different evolutionary pressures apply to the function of the individual GRKs.
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Phosphatidylinositol 4,5-Bisphosphate (PIP2)-enhanced G Protein-coupled Receptor Kinase (GRK) Activity: LOCATION, STRUCTURE, AND REGULATION OF THE PIP2 BINDING SITE DISTINGUISHES THE GRK SUBFAMILIES
Journal of Biological Chemistry, 1996Co-Authors: J A Pitcher, Walter J. Koch, Richard T Premont, Zl Fredericks, W C Stone, Robert H. Stoffel, Robert J LefkowitzAbstract:Abstract The G protein-coupled receptor kinases (GRKs) phosphorylate agonist occupied G protein-coupled receptors and play an important role in mediating receptor desensitization. The localization of these enzymes to their membrane incorporated substrates is required for their efficient function and appears to be a highly regulated process. In this study we demonstrate that phosphatidylinositol 4,5-bisphosphate (PIP2) enhances GRK5-mediated β-adrenergic receptor (βAR) phosphorylation by directly interacting with this enzyme and facilitating its membrane association. GRK5-mediated phosphorylation of a soluble peptide substrate is unaffected by PIP2, suggesting that the PIP2-enhanced receptor kinase activity arises as a consequence of this membrane localization. The lipid binding site of GRK5 exhibits a high degree of specificity and appears to reside in the amino terminus of this enzyme. Mutation of six basic residues at positions 22, 23, 24, 26, 28, and 29 of GRK5 ablates the ability of this kinase to bind PIP2. This region of the GRK5, which has a similar distribution of basic amino acids to the PIP2 binding site of gelsolin, is highly conserved between members of the GRK4 subfamily (GRK4, GRK5, and GRK6). Indeed, all the members of the GRK4 subfamily exhibit PIP2-dependent receptor kinase activity. We have shown previously that the membrane association of βARK (β-adrenergic receptor kinase) (GRK2) is mediated, in vitro, by the simultaneous binding of PIP2 and the βγ subunits of heterotrimeric G proteins to the carboxyl-terminal pleckstrin homology domain of this enzyme (Pitcher, J. A., Touhara, K., Payne, E. S., and Lefkowitz, R. J. (1995) J. Biol. Chem.270, 11707-11710). Thus, five members of the GRK family bind PIP2, βARK (GRK2), βARK2 (GRK3), GRK4, GRK5, and GRK6. However, the structure, location, and regulation of the PIP2 binding site distinguishes the βARK (GRK2 and GRK3) and GRK4 (GRK4, GRK5, and GRK6) subfamilies.
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Monoclonal antibodies reveal receptor specificity among G-protein-coupled receptor kinases.
Proceedings of the National Academy of Sciences of the United States of America, 1996Co-Authors: Martin Oppermann, María A. Diversé-pierluissi, Mark H. Drazner, Sara L. Dyer, Neil J. Freedman, Karsten Peppel, Robert J LefkowitzAbstract:Abstract Guanine nucleotide-binding regulatory protein (G protein)-coupled receptor kinases (GRKs) constitute a family of serine/threonine kinases that play a major role in the agonist-induced phosphorylation and desensitization of G-protein-coupled receptors. Herein we describe the generation of monoclonal antibodies (mAbs) that specifically react with GRK2 and GRK3 or with GRK4, GRK5, and GRK6. They are used in several different receptor systems to identify the kinases that are responsible for receptor phosphorylation and desensitization. The ability of these reagents to inhibit GRK- mediated receptor phosphorylation is demonstrated in permeabilized 293 cells that overexpress individual GRKs and the type 1A angiotensin II receptor. We also use this approach to identify the endogenous GRKs that are responsible for the agonist-induced phosphorylation of epitope-tagged beta2- adrenergic receptors (beta2ARs) overexpressed in rabbit ventricular myocytes that are infected with a recombinant adenovirus. In these myocytes, anti-GRK2/3 mAbs inhibit isoproterenol-induced receptor phosphorylation by 77%, while GRK4-6-specific mAbs have no effect. Consistent with the operation of a betaAR kinase-mediated mechanism, GRK2 is identified by immunoblot analysis as well as in a functional assay as the predominant GRK expressed in these cells. Microinjection of GRK2/3-specific mAbs into chicken sensory neurons, which have been shown to express a GRK3-like protein, abolishes desensitization of the alpha2AR-mediated calcium current inhibition. The intracellular inhibition of endogenous GRKs by mAbs represents a novel approach to the study of receptor specificities among GRKs that should be widely applicable to many G-protein-coupled receptors.
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Members of the G protein-coupled receptor kinase family that phosphorylate the beta2-adrenergic receptor facilitate sequestration.
Biochemistry, 1996Co-Authors: Luc Ménard, Stephen S. G. Ferguson, Richard T Premont, Robert J Lefkowitz, Larry S. Barak, Lucie Bertrand, Anne-marie Colapietro, Marc G CaronAbstract:We recently reported that a β2-adrenergic receptor (β2AR) mutant, Y326A, defective in its ability to sequester in response to agonist stimulation was a poor substrate for G protein-coupled receptor kinase (GRK)-mediated phosphorylation; however, its ability to be phosphorylated and sequestered could be restored by overexpressing GRK2 [Ferguson et al. (1995) J. Biol. Chem. 270, 24782]. In the present report, we tested the ability of each of the known GRKs (GRK1−6) to phosphorylate and rescue the sequestration of the Y326A mutant in HEK-293 cells. We demonstrate that in addition to GRK2, GRK3−6 can phosphorylate the Y326A mutant and rescue its sequestration; however, GRK1 was totally ineffective in rescuing either the phosphorylation or the sequestration of the mutant receptor. We found that the agonist-dependent rescue of Y326A mutant phosphorylation by GRK2, -3, and -5 was associated with the agonist-dependent rescue of sequestration. In contrast, overexpression of GRK4 and -6 led mainly to agonist-indepe...
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characterization of the g protein coupled receptor kinase GRK4 identification of four splice variants
Journal of Biological Chemistry, 1996Co-Authors: Richard T Premont, Alexander D Macrae, Robert H. Stoffel, Julie A. Pitcher, Namjin Chung, Christine Ambrose, James Inglese, Marcy E Macdonald, Robert J LefkowitzAbstract:A novel human G protein-coupled receptor kinase was recently identified by positional cloning in the search for the Huntington's disease locus (Ambrose, C., James, M., Barnes, G., Lin, C., Bates, G., Altherr, M., Duyao, M., Groot, N., Church, D., Wasmuth, J. J., Lehrach, H., Housman, D., Buckler, A., Gusella, J. F., and MacDonald, M. E. (1993) Hum. Mol. Genet. 1, 697-703). Comparison of the deduced amino acid sequence of GRK4 with those of the closely related GRK5 and GRK6 suggested the apparent loss of 32 codons in the amino-terminal domain and 46 codons in the carboxyl-terminal domain of GRK4. These two regions undergo alternative splicing in the GRK4 mRNA, resulting from the presence or absence of exons filling one or both of these apparent gaps. Each inserted sequence maintains the open reading frame, and the deduced amino acid sequences are similar to corresponding regions of GRK5 and GRK6. Thus, the GRK4 mRNA and the GRK4 protein can exist as four distinct variant forms. The human GRK4 gene is composed of 16 exons extending over 75 kilobase pairs of DNA. The two alternatively spliced exons correspond to exons II and XV. The genomic organization of the GRK4 gene is completely distinct from that of the human GRK2 gene, highlighting the evolutionary distance since the divergence of these two genes. Human GRK4 mRNA is expressed highly only in testis, and both alternative exons are abundant in testis mRNA. The four GRK4 proteins have been expressed, and all incorporate [3H]palmitate. GRK4 is capable of augmenting the desensitization of the rat luteinizing hormone/chorionic gonadotropin receptor upon coexpression in HEK293 cells and of phosphorylating the agonist-occupied, purified beta2-adrenergic receptor, indicating that GRK4 is a functional protein kinase.
