Protein-coupled Receptor Kinase Type Research Articles

Caveolin-1, a Determinant of the Fate of MCF-7 Breast Cancer Cells.

The scaffolding protein, caveolin-1 (Cav-1), participates in multiple cellular functions including promotion of sodium excretion from the kidney. Loss of expression of Cav-1 is associated with tumorigenesis of various types of cancer. We have shown the potential link between hypertension and breast cancer via abnormal function of the G protein-coupled receptor kinase type 4 (GRK4). The current studies tested the hypothesis that Cav-1 acts as a tumor-suppressive factor in breast cancer cells and enhances the sensitivity to the inhibitory effect of the type 1 dopaminergic receptor (D1R). Michigan Cancer Foundation (MCF) MCF-7 cells stably expressing a Cav-1/mCherry fusion protein or mCherry alone were used as models to examine the effect of Cav-1 on cell growth, apoptosis, and senescence. Cell proliferation was determined by cell counting, cell cycle analysis (flow cytometry), and BrdU incorporation. Apoptosis was determined using the Cell Death Detection ELISA kit from Roche Diagnosis. Senescence was determined using the senescence associated beta galactosidase (SA-β-gal) assay. Reactive oxygen species (ROS) was measured using 2',7'-dichlorodihydrofluorescein diacetate. Western blot analysis was used to measure activation of signaling pathway molecules. All statistical analyses were conducted with Microsoft Excel. Overexpression of Cav-1 in MCF-7 cells reduced cellular growth rate. Both inhibition of proliferation and induction of cell death are contributing factors. Multiple signaling pathways were activated in Cav-1-expressing MCF-7 cells. Activation of Akt was prominent. In MCF-7-expressing Cav-1 (MCF-7 Cav-1) cells, the levels of phosphorylated Akt at S473 and T308 were increased 28- and 8.7-fold, respectively. Instead of protecting cells from apoptosis, extremely high levels of activated Akt resulted in increased levels of ROS which led to apoptosis and senescence. The tumor-suppressive effect plus downregulation of GRK4 makes Cav-1-expressing MCF-7 cells significantly more sensitive to the inhibitory effect of the D1R agonist, SKF38393. Caveolin-1 acts as a tumor-suppressing factor via extreme activation of Akt and down regulation of survival factors such as GRK4, survivin, and cyclin D1.

ZBTB16 eases lipopolysaccharide‑elicited inflammation, apoptosis and degradation of extracellular matrix in chondrocytes during osteoarthritis by suppressing GRK2 transcription.

Osteoarthritis (OA) is a chronic degenerative disease of the bone that is a major contributor of disability in the elderly population. Zinc finger and BTB domain-containing 16 (ZBTB16) is a transcription factor that has been previously revealed to be impaired in human OA tissues. The present study was designed to elaborate the potential impact of ZBTB16 on OA and to possibly assess any latent regulatory mechanism. ZBTB16 expression in human OA tissues was examined using the Gene Expression Series (GSE) database (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE169077) whereas ZBTB16 expression in chondrocytes was examined using reverse transcription-quantitative PCR (RT-qPCR) and western blotting. Cell viability was examined using a Cell Counting Kit-8 assay. A TUNEL assay and western blotting were used to assess cell apoptosis and apoptosis-related markers, including Bcl-2, Bax and cleaved caspase-3. The levels and expression of inflammatory factors, including TNF-α, IL-1β and IL-6, were determined by ELISA and western blotting. RT-qPCR and western blotting were also used to analyze the expression levels of extracellular matrix (ECM)-degrading enzymes, including MMP-13, a disintegrin-like and metalloproteinase with thrombospondin type-1 motifs-5, aggrecan and collagen type II α1. After the potential binding of ZBTB16 with the G protein coupled receptor kinase type 2 (GRK2) promoter was predicted using the Cistrome DB database, GRK2 expression was confirmed by RT-qPCR and western blotting. Chromatin immunoprecipitation and luciferase reporter assays were then used to determine the potential interaction between ZBTB16 and the GRK2 promoter. Following GRK2 overexpression in ZBTB16-overexpressing chondrocytes by co-transfection of GRK2 and ZBTB16 overexpression plasmids, the aforementioned functional experiments were performed again. ZBTB16 expression was found to be reduced in human OA tissues compared with in normal cartilage tissues and lipopolysaccharide (LPS)-stimulated chondrocytes. ZBTB16 overexpression increased cell viability whilst decreasing apoptosis, inflammation and ECM degradation by LPS-treated chondrocytes. In addition, GRK2 expression was found to be increased in LPS-stimulated chondrocytes. ZBTB16 successfully bound to the GRK2 promoter, which negatively modulated GRK2 expression. GRK2 upregulation reversed the effects of ZBTB16 overexpression on the viability, apoptosis, inflammation and ECM degradation by LPS-challenged chondrocytes. In conclusion, these data suggest that ZBTB16 may inhibit the development of OA through the transcriptional inactivation of GRK2.

The Metabolic Role of GRK2 in Insulin Resistance and Associated Conditions.

Insulin resistance (IRES) is a pathophysiological condition characterized by the reduced response to insulin of several tissues, including myocardial and skeletal muscle. IRES is associated with obesity, glucose intolerance, dyslipidemia, and hypertension, evolves toward type 2 diabetes, and increases the risk of developing cardiovascular diseases. Several studies designed to explore the mechanisms involved in IRES allowed the identification of a multitude of potential molecular targets. Among the most promising, G Protein Coupled Receptor Kinase type 2 (GRK2) appears to be a suitable one given its functional implications in many cellular processes. In this review, we will discuss the metabolic role of GRK2 in those conditions that are characterized by insulin resistance (diabetes, hypertension, heart failure), and the potentiality of its inhibition as a therapeutic strategy to revert both insulin resistance and its associated phenotypes.

Molecular mechanisms underlying the GRK4 65L‐mediated hypertension in mice

The genetic causes of salt sensitivity andhypertension in humans are not completely understood. The kidney plays a preeminent regulatory rolein blood pressure (BP) homeostasis and water and electrolyte balance. The renaldopamine receptors, D1R and D3R, engender natriuresis viathe inhibition of renal Na+ transport, whereas the angiotensin IItype 1 receptor (AT1R) does the opposite. The renal paracrineinhibition of Na+ transport by dopamine is impaired insalt‐sensitive rats, mice, and humans. Agonist activation promotes thephosphorylation of D1R and D3R by the G protein‐coupledreceptor kinase type 4 (GRK4), whose gene variants impair D1R and D3Ractivity. The global expression of GRK4 65R>L in transgenic mice results in salt‐sensitive hypertension, in part, due toincreased endogenous GRK4 and AT1R expression. To demonstrate thespecific renal causal mechanisms in GRK4 65R>L‐mediated hypertension, weheterologously expressed the GRK4 65R>Lvs. GRK4 wild‐type (WT) transgenes in the kidneys of Grk4 knockout mice on normal salt diet. The transgenes were delivered selectively into the renal tubules by thebilateral retrograde ureteral infusion of AAV‐9 vectors. The expression anddistribution along the entire nephron of the GRK4 WT and GRK4 65R>L were similar in both groups. However, the renal tubule‐restricted expression ofGRK4 65R>L increased the BP (117±4vs. 93±1 mm Hg, P<0.05, n=4), while that of the GRK4 WT only tendedto increase the BP (105±6 vs. 96±2 mmHg, n=5), indicating that the presence of the GRK4 variant in the kidney causedthe increase in BP. We next evaluated the renal expression profiles of selectgenes. We found that the pro‐natriuretic D1R (0.81±0.01 vs. 1.28±0.04, P<0.01) and D3R(0.44±0.02 vs. 1.27±0.07, P<0.01)were decreased whereas the anti‐natriuretic Na+/K+‐ ATPase(1.14±0.024 vs. 1.0 ±0.007, P<0.05) and α‐ENaC (1.4±0.14 vs. 1.0±0.11, P<0.05) were increased, demonstrating themechanistic changes that underlie the hypertension in these mice. Interestingly, we also observed that the proximal tubule Na+transporters NaPi2 (0.81±0.02 vs. 1.04±0.02), SGLT2 (0.89±0.03 vs. 1.07±0.05), NBCe2(0.50±0.07 vs. 1.15±0.03), and the AT1R(0.82±0.02 vs 1.02±0.02) were decreased, which may represent insufficientcompensatory mechanisms against the increase in BP. Our results highlight theunderlying and compensatory renal mechanisms for the hypertension thatdeveloped in mice with either kidney‐restricted or globally expressed GRK4 65R>L.Support or Funding InformationR01DK039308, R01HL092196, and P01HL074940This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Abstract 115: Renal-Specific Expression of Human Grk4 R65L in Mice Lacking Grk4 Increases Blood Pressure

The genetic causes of salt (NaCl) sensitivity in humans are not well understood. The kidney is critical to the overall fluid and electrolyte balance and long-term regulation of blood pressure (BP). Therefore, the pathogenesis of salt sensitivity must involve a deficient renal NaCl handling. The renal paracrine inhibition of Na + transport by dopamine is impaired in salt-sensitive rats, mice, and humans due to decreased dopamine D1 receptor (D1R) function and not related to a primary defect of the D1R gene but rather due to its uncoupling from second messengers caused by activating variants of the human G protein-coupled receptor kinase type 4 (h GRK4 ) (R65L, A142V, and A486V). We have shown that global transgenic mice expressing hGRK4 R65L variant have salt-sensitive hypertension. To determine the role of the kidney in the salt-sensitivity of these mice, we developed a mouse model of kidney-specific expression of the R65L variant (KS hGRK4 R65L) by bilateral ureteral infusion of AAV vectors carrying the variant in GRK4 knockout mice. Mice infused with hGRK4 wild-type served as controls (KS hGRK4 WT). Systolic BP (SBP) was measured under anesthesia in the mice before and after infusion of the AAV vectors. SBP before AAV was similar in both groups. SBP post AAV increased in KS hGRK4 R65L (93±1 vs 117±4 mmHg, P<0.05, n=4) but not in KS hGRK4 WT (96±2 vs 105±6, n=5). Renal mRNA expressions of dopamine 1 (D1R, 0.81±0.01 vs 1.28±0.04, P<0.01), D3R (0.44±0.02 vs 1.27±0.07, P<0.01) and angiotensin AT1A (AT1A, 0.78±0.02 vs 1.02±0.02, P<0.01) receptors were decreased in renal cortex of KS hGRK4 R65L relative to KS hGRK4 WT. In addition, the expressions of Na + /K + ATPase (1.12±0.03 vs 1.0 ±0.007, P<0.05) and ENaC (1.4±0.14 vs 1.0±0.11, P<0.05) were increased while those of the proximal tubule Na + transporters NaPi2 (0.81±0.02 vs 1.04±0.02), SGLT2 (0.89±0.03 vs 1.07±0.05), and NBCe2 (0.50±0.07 vs 1.15±0.03) were decreased. Our results show that kidney specific expression of hGRK4 R65L increased SBP and produced changes in the expression of receptors and Na + transporters, pump, and channel but their roles in the increased SBP remain to be determined.

In Utero Exposure to Fine Particulate Matter Causes Hypertension Due to Impaired Renal Dopamine D1 Receptor in Offspring

Background/Aims: Adverse environment in utero can modulate adult phenotypes including blood pressure. Fine particulate matter (PM_2.5) exposure in utero causes hypertension in the offspring, but the exact mechanisms are not clear. Renal dopamine D₁ receptor (D₁R), regulated by G protein-coupled receptor kinase type 4 (GRK4), plays an important role in the regulation of renal sodium transport and blood pressure. In this present study, we determined if renal D₁R dysfunction is involved in PM_2.5–induced hypertension in the offspring. Methods: Pregnant Sprague–Dawley rats were given an oropharyngeal drip of PM_2.5 (1.0 mg/kg) at gestation day 8, 10, and 12. The blood pressure, 24-hour sodium excretion, and urine volume were measured in the offspring. The expression levels of GRK4 and D₁R were determined by immunoblotting. The phosphorylation of D₁R was investigated using immunoprecipitation. Plasma malondialdehyde and superoxide dismutase levels were also measured in the offspring. Results: As compared with saline-treated dams, offspring of PM_2.5-treated dams had increased blood pressure, impaired sodium excretion, and reduced D₁R-mediated natriuresis and diuresis, accompanied by decreased renal D₁R expression and GRK4 expression. The impaired renal D₁R function and increased GRK4 expression could be caused by increased reactive oxidative stress (ROS) induced by PM_2.5 exposure. Administration of tempol, a redox-cycling nitroxide, for 4 weeks in the offspring of PM_2.5-treated dam normalized the decreased renal D₁R expression and increased renal D₁R phosphorylation and GRK4 expression. Furthermore, tempol normalized the increased renal expression of c-Myc, a transcription factor that regulates GRK4 expression. Conclusions: In utero exposure to PM_2.5 increases ROS and GRK4 expression, impairs D₁R-mediated sodium excretion, and increases blood pressure in the offspring. These studies suggest that normalization of D₁R function may be a target for the prevention and treatment of the hypertension in offspring of mothers exposed to PM_2.5 during pregnancy.

Downregulation of Renal G Protein-Coupled Receptor Kinase Type 4 Expression via Ultrasound-Targeted Microbubble Destruction Lowers Blood Pressure in Spontaneously Hypertensive Rats.

BackgroundG protein–coupled receptor kinase type 4 (GRK4) plays a vital role in the long‐term control of blood pressure (BP) and sodium excretion by regulating renal G protein–coupled receptor phosphorylation, including dopamine type 1 receptor (D1R). Ultrasound‐targeted microbubble destruction (UTMD) is a promising method for gene delivery. Whether this method can deliver GRK4 small interfering RNA (siRNA) and lower BP is not known.Methods and ResultsBP, 24‐hour sodium excretion, and urine volume were measured after UTMD‐targeted GRK4 siRNA delivery to the kidney in spontaneously hypertensive rats. The expression levels of GRK4 and D1R were determined by immunoblotting. The phosphorylation of D1R was investigated using immunoprecipitation. The present study revealed that UTMD‐mediated renal GRK4 siRNA delivery efficiently reduced GRK4 expression and lowered BP in spontaneously hypertensive rats, accompanied by increased sodium excretion. The increased sodium excretion might be accounted for by the UTMD regulation of D1R phosphorylation and function in spontaneously hypertensive rats. Further analysis showed that, although UTMD had no effect on D1R expression, it reduced D1R phosphorylation in spontaneously hypertensive rats kidneys and consequently increased D1R‐mediated natriuresis and diuresis.ConclusionsTaken together, these study results indicate that UTMD‐targeted GRK4 siRNA delivery to the kidney effectively reduces D1R phosphorylation by inhibiting renal GRK4 expression, improving D1R‐mediated natriuresis and diuresis, and lowering BP, which may provide a promising novel strategy for gene therapy for hypertension.

Integrating GRK2 and NFkappaB in the Pathophysiology of Cardiac Hypertrophy.

G protein coupled receptor kinase type 2 (GRK2) plays an important role in the development and maintenance of cardiac hypertrophy and heart failure even if its exact role is still unknown. In this study, we assessed the effect of GRK2 on the regulation of cardiac hypertrophy. In H9C2 cells, GRK2 overexpression increased atrial natriuretic factor (ANF) activity and enhanced phenylephrine-induced ANF response, and this is associated with an increase of NFκB transcriptional activity. The kinase dead mutant and a synthetic inhibitor of GRK2 activity exerted the opposite effect, suggesting that GRK2 regulates hypertrophy through upregulation of NFκB activity in a phosphorylation-dependent manner. In two different in vivo models of left ventricle hypertrophy (LVH), the selective inhibition of GRK2 activity prevented hypertrophy and reduced NFκB transcription activity. Our results suggest a previously undisclosed role for GRK2 in the regulation of hypertrophic responses and propose GRK2 as potential therapeutic target for limiting LVH.

Prenatal lipopolysaccharide exposure results in dysfunction of the renal dopamine D1 receptor in offspring

Adverse environment in early life can modulate the adult phenotype, including blood pressure. Lipopolysaccharide (LPS) exposure in utero results in increased blood pressure in the offspring, but the exact mechanisms are not clear. Studies have shown that the renal dopamine D1 receptor (D1R) plays an important role in maintaining sodium homeostasis and normal blood pressure; dysfunction of D1R is associated with oxidative stress and hypertension. In this study, we determined if dysfunction of the renal D1R is involved in fetal-programmed hypertension, and if oxidative stress contributes to this process. Pregnant Sprague–Dawley (SD) rats were intraperitoneally injected with LPS (0.79mg/kg) or saline at gestation days 8, 10, and 12. As compared with saline-injected (control) dams, offspring of LPS-treated dams had increased blood pressure, decreased renal sodium excretion, and increased markers of oxidative stress. In addition, offspring of LPS-treated dams had decreased renal D1R expression, increased D1R phosphorylation, and G protein-coupled receptor kinase type 2 (GRK2) and type 4 (GRK4) protein expression, and impaired D1R-mediated natriuresis and diuresis. All of the findings in the offspring of LPS-treated dams were normalized after treatment with TEMPOL, an oxygen free radical scavenger. In conclusion, prenatal LPS exposure, via an increase in oxidative stress, impairs renal D1R function and leads to hypertension in the offspring. Normalization of renal D1R function by amelioration of oxidative stress may be a therapeutic target of fetal programming of hypertension.

Abstract 517: Ubiquitylation of G Protein Coupled Receptor Kinase 4 (GRK4) Regulates its Localization and Abundance

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<0.05, N=3) as well as a mCherry GRK4 fusion construct (6.98±0.50 fold increase over VEH: VEH 5,044±3229 RFU; CLBL 35,210±2547 RFU, P<0.05, N=3). The ubiquitin binding sites (AA 216,217; MYACK 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<0.01, N=12). In summary, GRK4 expression levels are negatively regulated by the ubiquitin proteasome system and ubiquitylation of GRK4 prevents nuclear import. We hypothesize that ubiquitylation is sterically hindering the nuclear localization sequence in GRK4 from interacting with the nuclear import machinery of the cell. Through manipulation of GRK4 ubiquitylation, one may be able to separate nuclear vs. cytoplasmic activity of GRK4 and reverse the negative impact overactive GRK4 has on dopaminergic signaling.

Abstract 38: Exosomes Secreted From Human Renal Proximal Tubule Cells Can Transfer Their Contents to Proximal Tubule, Distal Tubule and Collecting Duct Cells

Exosomes are 50-90 nm extracellular membrane particles that may mediate trans-cellular communication between cells and tissues. We have previously found that urinary exosomes contain miRNA that are biomarkers for salt sensitivity and inverse salt sensitivity of blood pressure. We also found that urinary exosomes contain dopamine 1 receptor and G protein-coupled receptor kinase type 4. We have also shown that exosomes can transfer between cultured human renal proximal tubule cells (RPTC). Here, we further explored RPTC-to-RPTC exosomal transfer using 5 RPTC lines containing exosomes that were fluorescently labelled with either exosomal-specific marker CD63-EGFP or CD9-mCherry. Transfer between RPTCs was confirmed by co-culturing CD63-EGFP and CD9-mCherry stable RPTC clones and performing live confocal microscopy. Dual color time-lapse videos reveal unique cellular secretion and uptake patterns for exosomes and eventual accumulation into the multi-vesicular body. We hypothesized that proximal tubule-derived exosomes could also be taken up by the distal tubule (DT). Five DT and 3 collecting duct (CD) cell lines were isolated and immortalized by infecting with hTert-containing lentiviral particles. DT cells were isolated from human kidney by collagenase digestion and biotinylated lectin or immuno-magnetic affinity separation (using dolichos biflorus agglutinin (DBA) or anti-Tamm-Horsfall protein antibody). CD cells were similarly isolated with anti-L1CAM antibody. Using sterile-filtered cell culture supernatant from 3 different CD9-mCherry RPTC lines, all 5 DT cell lines showed exosome transfer at 24 hours as measured by microplate fluorometry (10,406 ± 1132 SEM RFU above background, p<0.0001, n=30). Similarly, all 3 CD cell lines showed significant exosome transfer (8773 ± 1558 SEM RFU, p<0.0001, n=18). CD63-EGFP exosomes from 2RPTC lines also transferred to all of the DT cell lines (1713 ± 1131 RFU above background, p<0.0001, n=30) and CD cell lines (1007 ± 315 RFU, p<0.01, n=18). In conclusion, intra-nephron cellular exosomal transfer is demonstrated in RPTCs and between RPTCs and distal tubule and collecting duct cells. This proximal to distal nephron transfer may represent a new trans-cellular communication within the human kidney.

Abstract 616: Caveolin-1 (CAV1) inhibits G Protein Coupled Receptor Kinase Type 4 (GRK4) kinase activity

In human renal proximal tubule cells, the dopamine-1 receptor (D1R) has been shown to increase cAMP production and negatively regulate sodium transport. Defects in D1R signaling have been associated with SNPs in G-protein-coupled receptor kinase type 4 (GRK4) which hyperphosphorylates and decreases D1R function, and the D1R uncoupling phenotype is fully reverted by reducing GRK4 expression with siRNA. We have also shown that CAV1 co-immunoprecipitates with GRK4, and that the normal D1R inhibition of the sodium transporter, NaKATPase alpha, can be blocked using siRNA to CAV1. We therefore hypothesized that CAV1 directly physically interacts with GRK4 and inhibits its kinase activity. Using purified human GRK4 and CAV1 proteins and an in-vitro peptide homogeneous kinase assay, we investigated the effects of caveolin-1 (CAV1) on the kinase activity of GRK4. By overexpressing and isolating a tandem affinity purified protein (TAP tagged) functional version of human CAV1 isolated from a human renal proximal tubule cell line, we were able to ensure that all of the post-translational modifications of the protein were present. Utilizing a peptide Ser/Thr kinase activity assay, we demonstrated that with an excess of CAV1, the kinase activity of GRK4 is inhibited by 37.8±1.3% (n=4, p<0.05). Using this same assay, we showed that purified human calcium calmodulin 1 (CALM1), a known inhibitor of GRK4 when using a complete cell lysate, does not significantly decrease the kinase activity of purified GRK4 using a CALM1 concentration over 100 times the published Kd value. This suggests that there is a missing co-factor or factors required for CALM1 to inhibit GRK4. In summary, we report for the first time that CAV1 inhibits the kinase activity of GRK4, and that a known inhibitor of GRK4, CALM1, does not directly inhibit GRK4 kinase activity using purified proteins in an in-vitro kinase activity assay.

Corrections

HomeHypertensionVol. 56, No. 5Corrections Free AccessCorrectionPDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessCorrectionPDF/EPUBCorrections Originally published1 Nov 2010https://doi.org/10.1161/HYP.0b013e3181f914eeHypertension. 2010;56:e167This article corrects the followingHK-2 Human Renal Proximal Tubule Cells as a Model for G Protein–Coupled Receptor Kinase Type 4–Mediated Dopamine 1 Receptor UncouplingIn the Hypertension article by Gildea et al (Gildea JJ, Shah I, Weiss R, Casscells ND, McGrath HE, Zhang J, Felder RA. HK-2 Human Renal Proximal Tubule Cells as a Model for G Protein–Coupled Receptor Kinase Type 4–Mediated Dopamine 1 Receptor Uncoupling. Hypertension. 2010;56: 505–511), author John E. Jones was erroneously omitted from the author line. The corrected author listing is as follows: John J. Gildea, Ishan Shah, Ryan Weiss, Nicholas D. Casscells, Helen E. McGrath, Jin Zhang, John E. Jones, Robin A. Felder.The authors regret the error.This correction has been made to the current online version of the article, which is available at http://hyper.ahajournals.org/cgi/content/full/56/3/505. Previous Back to top Next FiguresReferencesRelatedDetailsRelated articlesHK-2 Human Renal Proximal Tubule Cells as a Model for G Protein–Coupled Receptor Kinase Type 4–Mediated Dopamine 1 Receptor UncouplingJohn J. Gildea, et al. Hypertension. 2010;56:505-511 November 2010Vol 56, Issue 5 Advertisement Article InformationMetrics https://doi.org/10.1161/HYP.0b013e3181f914ee Originally publishedNovember 1, 2010 PDF download Advertisement

HK-2 Human Renal Proximal Tubule Cells as a Model for G Protein–Coupled Receptor Kinase Type 4–Mediated Dopamine 1 Receptor Uncoupling

HK-2 human renal proximal tubule cells (RPTC) are commonly used in the in vitro study of "normal" RPTCs. We discovered recently that HK-2 cells are uncoupled from dopamine 1 receptor (D(1)R) adenylyl cyclase (AC) stimulation. We hypothesized that G protein-coupled receptor kinase type 4 (GRK4) single nucleotide polymorphisms may be responsible for the D(1)R/AC uncoupling in HK-2. This hypothesis was tested by genotyping GRK4 single nucleotide polymorphisms, measuring D(1)-like receptor agonist (fenoldopam)-stimulated cAMP accumulation, quantifying D(1)R inhibition of sodium transport, and testing the ability of GRK4 small interfering RNA to reverse the D(1)R/AC uncoupling. We compared HK-2 with 2 normally coupled human RPTC cell lines and 2 uncoupled RPTC cell lines. The HK-2 cell line was found to have 4 of 6 potential GRK4 single nucleotide polymorphisms known to uncouple the D(1)R from AC (namely, R65L, A142V, and A486V). AC response to fenoldopam stimulation was increased in the 2 normally coupled human RPTC cell lines (FEN: 2.02+/-0.05-fold and 2.33+/-0.19-fold over control; P<0.001; n=4) but not in the 2 uncoupled or HK-2 cell lines. GRK4 small interfering RNA rescued the fenoldopam-mediated AC stimulation in the uncoupled cells, including HK-2. The expected fenoldopam-mediated inhibition of sodium hydrogen exchanger type 3 was absent in HK-2 (n=6) and uncoupled RPTC cell lines (n=6) but was observed in the 2 normally coupled human RPTC cell lines (-25.41+/-4.7% and -27.36+/-2.70%; P<0.001; n=6), which express wild-type GRK4. Despite the fact that HK-2 cells retain many functional characteristics of RPTCs, they are not normal from the perspective of dopaminergic function.

Overexpression of β-Arrestin 2 in Peripheral Blood Mononuclear Cells of Patients With Cryptococcal Meningitis

Cryptococcal meningitis is often associated with elevated IL-10 levels, which suggest a dysregulation in the antifungal immune response. beta-Arrestin 2 plays a major role in desensitization of G-protein-coupled receptors involved in the immune responses, provides a scaffolding platform for modification of many signal transduction proteins, and binds Src and MAP kinases family members. This study compared the levels of beta-arrestin 2 mRNA and protein in peripheral blood mononuclear cells (PBMC) of patients with cryptococcal meningitis detected. The interferon-gamma (IFN-gamma) serum concentration was determined with enzyme-linked immunosorbent assay (ELISA) to reveal its relationship with beta-arrestin 2. The effect of modulation of beta-arrestin 2 on cytotoxic activity against Cryptococcus was explored via transfection and interference of beta-arrestin 2. PBMCs of patients with cryptococcal meningitis exhibited significantly elevated levels of beta-arrestin 2 and a positive correlation between beta-arrestin 2 and IL-10 levels existed in patients' serum, but a negative correlation was found between beta-arrestin 2 and IFN-gamma expression. In conclusion, elevated expression of beta-arrestin 2 in PBMCs of patients with cryptococcal meningitis correlated with a reduced cytotoxic activity against Cryptococcus. This study suggests that reduced beta-arrestin 2 mRNA levels or inhibition of beta-arrestin 2 activity may augment INF-gamma production, and ultimately, the anti-Cryptococcus immune response of infected patients.

Abstract 5413: G Protein-Coupled Receptor Type 4 Gene Variants and Response to Antihypertensive Medication

Renal dopamine counteracts the prohypertensive and sodium retaining actions of the renin-angiotensin system (RAS). In genetic hypertension, the ability of dopamine to counteract the actions of the RAS is impaired by an increased constitutive activity of genetic variants of G protein-coupled receptor kinase type 4 (GRK4) that desensitize DRD1 dopamine receptors. Selective renal silencing of Grk4 improves DRD1 function and ameliorates the hypertension in spontaneously hypertensive rats. GRK4 γ wild-type transgenic mice are normotensive and salt-resistant; GRK4 γ 142V transgenic mice are hypertensive while Grk4 γ 486V transgenic mice are salt-sensitive. Renal Grk4 and angiotensin type 1 receptor (AT1R) interact to regulate blood pressure (BP) in rats and GRK4 γ 142V transgenic mice. We tested the association between essential hypertension in 1102 Japanese (616 cases and 486 controls) and single nucleotide polymorphisms (SNPs)/markers in the genes encoding GRK4, ACE, angiotensinogen, AT1R, aldosterone synthase, plasminogen activator inhibitor-1, DRD1, and GNB3. In addition, GRK4 genotype and the response to angiotensin receptor blockers (ARBs) without or with diuretic therapy in 161 Japanese with mild essential hypertension (systolic[SBP]/diastolic [DBP]=148.5±3.3/92.9±2.9 mmHg, age 55±10 yrs) were examined. All three GRK4 SNPs (R65L, A142V, and A486V) associated with hypertension (allelic and genotypic tests, P&lt;4×10 −8 for all three markers). The GRK4 three variant haplotype odds ratio for hypertension was 2.49(P&lt;4×10 −14 ). In the responders to ARBs (n=67) BP decreased to 132.8±3.7/84.7±3.1 mmHg and was highly associated with the GRK4142V (P&lt;0.003). The absolute decrease in SBP and DBP in response to ARB was also associated with GRK4142V (P&lt;0.007). The addition of a diuretic (bumetanide) to the non-responders (n=94) decreased BP (127.8±5.0/84.4±2.7, n=47) which was associated with GRK4A486V (P&lt;0.005). The GRK4 variant 142V may be both strongly predisposing to essential hypertension and predictive of the initial response to ARB monotherapy while the GRK4 variant 486V may be predictive of the response to dual therapy with an ARB and a diuretic. Therefore, pharmacogenetics of GRK4 is important in guiding the therapy for hypertension.

Caveolin-1 and Dopamine-Mediated Internalization of NaKATPase in Human Renal Proximal Tubule Cells

In moderate sodium-replete states, dopamine 1-like receptors (D1R/D5R) are responsible for regulating >50% of renal sodium excretion. This is partly mediated by internalization and inactivation of NaKATPase, when associated with adapter protein 2. We used dopaminergic stimulation via fenoldopam (D1-like receptor agonist) to study the interaction among D1-like receptors, caveolin-1 (CAV1), and the G protein-coupled receptor kinase type 4 in cultured human renal proximal tubule cells (RPTCs). We compared 2 groups of RPTCs, 1 of cell lines that were isolated from normal subjects (nRPTCs) and a second group of cell lines that have D1-like receptors that are uncoupled (uncoupled RPTCs) from adenylyl cyclase second messengers. In nRPTCs, fenoldopam increased the plasma membrane expression of D1R (10.0-fold) and CAV1 (1.3-fold) and markedly decreased G protein-coupled receptor kinase type 4 by 94+/-8%; no effects were seen in uncoupled RPTCs. Fenoldopam also increased the association of adapter protein 2 and NaKATPase by 53+/-9% in nRPTCs but not in uncoupled RPTCs. When CAV1 expression was reduced by 86.0+/-8.5% using small interfering RNA, restimulation of the D1-like receptors with fenoldopam in nRPTCs resulted in only a 7+/-9% increase in association between adapter protein 2 and NaKATPase. Basal CAV1 expression and association with G protein-coupled receptor kinase type 4 was decreased in uncoupled RPTCs (58+/-5% decrease in association) relative to nRPTCs. We conclude that the scaffolding protein CAV1 is necessary for the association of D1-like receptors with G protein-coupled receptor kinase type 4 and the adapter protein 2-associated reduction in plasma membrane NaKATPase.

Amelioration of Genetic Hypertension by Suppression of Renal G Protein–Coupled Receptor Kinase Type 4 Expression

Abnormalities in D1 dopamine receptor function in the kidney are present in some types of human essential and rodent genetic hypertension. We hypothesize that increased activity of G protein-coupled receptor kinase type 4 (GRK4) causes the impaired renal D1 receptor function in hypertension. We measured renal GRK4 and D1 and serine-phosphorylated D1 receptors and determined the effect of decreasing renal GRK4 protein by the chronic renal cortical interstitial infusion (4 weeks) of GRK4 antisense oligodeoxynucleotides (As-Odns) in conscious- uninephrectomized spontaneously hypertensive rats (SHRs) and their normotensive controls, Wistar-Kyoto (WKY) rats. Basal GRK4 expression and serine-phosphorylated D1 receptors were &90% higher in SHRs than in WKY rats and were decreased to a greater extent in SHRs than in WKY rats with GRK4 As-Odns treatment. Basal renal D1 receptor protein was similar in both rat strains. GRK4 As-Odns, but not scrambled oligodeoxynucleotides, increased sodium excretion and urine volume, attenuated the increase in arterial blood pressure with age, and decreased protein excretion in SHRs, effects that were not observed in WKY rats. These studies provide direct evidence of a crucial role of renal GRK4 in the D1 receptor control of sodium excretion and blood pressure in genetic hypertension.

Single-Nucleotide Polymorphisms for Diagnosis of Salt-Sensitive Hypertension

Salt-sensitive (SS) hypertension affects >30 million Americans and is often associated with low plasma renin activity. We tested the diagnostic validity of several candidate genes for SS and low-renin hypertension. In Japanese patients with newly diagnosed, untreated hypertension (n = 184), we studied polymorphisms in 10 genes, including G protein-coupled receptor kinase type 4 (GRK4), some variations of which are associated with hypertension and impair D1 receptor (D1R)-inhibited renal sodium transport. We used the multifactor dimensionality reduction method to determine the genotype associated with salt sensitivity (> or =10% increase in blood pressure with high sodium intake) or low renin. To determine whether the GRK4 genotype is associated with impaired D1R function, we tested the natriuretic effect of docarpamine, a dopamine prodrug, in normotensive individuals with or without GRK4 polymorphisms (n = 18). A genetic model based on GRK4 R65L, GRK4 A142V, and GRK4 A486V was 94.4% predictive of SS hypertension, whereas the single-locus model with only GRK4 A142V was 78.4% predictive, and a 2-locus model of GRK4 A142V and CYP11B2 C-344T was 77.8% predictive of low-renin hypertension. Sodium excretion was inversely related to the number of GRK4 variants in hypertensive persons, and the natriuretic response to dopaminergic stimulation was impaired in normotensive persons having > or =3 GRK4 gene variants. GRK4 gene variants are associated with SS and low-renin hypertension. However, the genetic model predicting SS hypertension is different from the model for low renin, suggesting genetic differences in these 2 phenotypes. Like low-renin testing, screening for GRK4 variants may be a useful diagnostic adjunct for detection of SS hypertension.

Functional genomics of the dopaminergic system in hypertension

Abnormalities in dopamine production and receptor function have been described in human essential hypertension and rodent models of genetic hypertension. Under normal conditions, D(1)-like receptors (D(1) and D(5)) inhibit sodium transport in the kidney and intestine. However, in the Dahl salt-sensitive and spontaneously hypertensive rats (SHRs) and in humans with essential hypertension, the D(1)-like receptor-mediated inhibition of epithelial sodium transport is impaired because of an uncoupling of the D(1)-like receptor from its G protein/effector complex. The uncoupling is receptor specific, organ selective, nephron-segment specific, precedes the onset of hypertension, and cosegregates with the hypertensive phenotype. The defective transduction of the renal dopaminergic signal is caused by activating variants of G protein-coupled receptor kinase type 4 (GRK4: R65L, A142V, A486V). The GRK4 locus is linked to and GRK4 gene variants are associated with human essential hypertension, especially in salt-sensitive hypertensive subjects. Indeed, the presence of three or more GRK4 variants impairs the natriuretic response to dopaminergic stimulation in humans. In genetically hypertensive rats, renal inhibition of GRK4 expression ameliorates the hypertension. In mice, overexpression of GRK4 variants causes hypertension either with or without salt sensitivity according to the variant. GRK4 gene variants, by preventing the natriuretic function of the dopaminergic system and by allowing the antinatriuretic factors (e.g., angiotensin II type 1 receptor) to predominate, may be responsible for salt sensitivity. Subclasses of hypertension may occur because of additional perturbations caused by variants of other genes, the quantitative interaction of which may vary depending upon the genetic background.