G protein-coupled receptor 4 variants in human essential hypertension

Robin A. Felder*, Hironobu Sanada*†, Jing Xu‡, Pei-Ying Yu‡, Zheng Wang‡, Hidetsuna Watanabe*, Laureano D. Asico*, Wei Wang‡, Shaopeng Zheng‡, Ikuyo Yamaguchi‡, Scott M. Williams§, James Gainer¶, Nancy J. Brown¶, Debra Hazen-Martinʈ, Lee-Jun C. Wong**, Jean E. Robillard††, Robert M. Carey‡‡, Gilbert M. Eisner‡, and Pedro A. Jose‡

*Department of Pathology, University of Virginia Health Sciences Center, Charlottesville, VA 22908; ‡Department of Pediatrics and Physiology and Biophysics, Georgetown University Medical Center, Washington, DC 20007; §Department of Microbiology, Meharry Medical College, Nashville, TN 37208; ¶Department of Medicine and Pharmacology, Vanderbilt University Medical Center, Nashville, TN 37232; ʈDepartment of Pathology, Medical University of South Carolina, Charleston, SC 29403; **Institute for Molecular and Human Genetics, Georgetown University Medical Center, Washington, DC 20007; ††Department of Pediatrics, University of Michigan College of Medicine, Ann Arbor, MI 48109; and ‡‡Department of Medicine, University of Virginia Health Sciences Center, Charlottesville, VA 22908

Communicated by Maria Iandolo New, Weill Medical College of Cornell University, New York, NY, December 21, 2001 (received for review August 10, 2001) Essential hypertension has a heritability as high as 30–50%, but its abnormal renal sodium transporters (3, 8, 13, 17). Also, the genetic cause(s) has not been determined despite intensive inves- coding region of the D1 receptor is unchanged in hypertensive tigation. The renal dopaminergic system exerts a pivotal role in subjects (16), as well as in rodents with genetic hypertension maintaining fluid and electrolyte balance and participates in the (unpublished studies). pathogenesis of genetic hypertension. In genetic hypertension, the In renal proximal tubules from humans with essential hyper- ability of and D1-like agonists to increase urinary sodium tension and from rodents with genetic hypertension, the D1-like excretion is impaired. A defective coupling between the D1 dopa- receptor is uncoupled from its G protein͞effector mine receptor and the G protein͞effector enzyme complex in the complex (3, 7–10, 16). This uncoupling is thought to be the proximal tubule of the kidney is the cause of the impaired renal mechanism for the failure of dopamine to engender a natriuresis dopaminergic action in genetic rodent and human essential hy- in genetic hypertension (3, 5, 11–15). This mechanism is similar pertension. We now report that, in human essential hypertension, to but distinct from homologous desensitization (18, 19) because single nucleotide polymorphisms of a G protein-coupled receptor the uncoupling in hypertension is independent of renal dopa- kinase, GRK4␥, increase G protein-coupled receptor kinase (GRK) mine levels (3, 16, 20). Similarly, the uncoupling is not due to activity and cause the serine phosphorylation and uncoupling of heterologous desensitization because the responsiveness of other ͞ the D1 receptor from its G protein effector enzyme complex in the G protein-coupled receptors (e.g., parathyroid hormone, ␤- renal proximal tubule and in transfected Chinese hamster ovary adrenergic, and cholecystokinin receptors) remains intact in the cells. Moreover, expressing GRK4␥A142V but not the wild-type prehypertensive spontaneously hypertensive rat (3, 8, 21–23). gene in transgenic mice produces hypertension and impairs the G protein-coupled receptor (GRKs) have been im- diuretic and natriuretic but not the hypotensive effects of D1-like plicated in genetic and acquired hypertension because they agonist stimulation. These findings provide a mechanism for the D1 participate in the desensitization of G protein-coupled receptors, receptor coupling defect in the kidney and may explain the including D1 receptors. The GRK-mediated desensitization is inability of the kidney to properly excrete sodium in genetic caused, in part, by serine phosphorylation of the receptor (18, hypertension. 19). We have reported that basal serine-phosphorylated D1 receptor is increased in renal proximal tubules from genetically ong-term regulation of blood pressure is vested in the organ hypertensive rodents as well as from humans with essential Lresponsible for the control of body fluid volume, the kidney hypertension (3, 16). (1, 2). Dopamine facilitates the antihypertensive function of the The seven members of the GRK family are divided into three kidney because it is both vasodilatory and natriuretic (3). subfamilies: GRK1 and GRK7 belong to the ␤ Dopamine (produced by renal proximal tubules) via D1-like subfamily; GRK2 and GRK3 belong to the -adrenergic receptor receptors is responsible for over 50% of incremental sodium kinase subfamily; and GRK4, GRK5, and GRK6 belong to the excretion when sodium intake is increased (3–6). The paracrine͞ GRK4 subfamily (24, 25). GRK2 expression and activity are autocrine dopaminergic regulation of sodium excretion is me- increased in lymphocytes from patients with essential hyperten- diated by tubular but not by hemodynamic mechanisms (6). The sion (26) and genetically hypertensive rats (27). However, the ability of dopamine and D1-like agonists to decrease renal increase in GRK2 expression and activity in the spontaneously proximal tubular sodium reabsorption is impaired in genetic hypertensive rat followed the development of hypertension (27). rodent hypertension and human essential hypertension (3, 5, Moreover, we found no difference in the sequence of the coding 7–15). Indeed, the aberrant D1-like receptor function in the region of GRK2 between hypertensive and normotensive human kidney precedes and cosegregates with high blood pressure in subjects (unpublished data). It is possible that the increase in spontaneously hypertensive rats. In addition, disruption of the GRK2 activity in lymphocytes of hypertensive patients (26, 27) D1 receptor in mice produces hypertension (12, 13). The pivotal is secondary to the high blood pressure, as has been suggested role of dopamine in the excretion of sodium after increased for the increase in GRK5 activity and expression in rodents with sodium intake has led to the hypothesis that an aberrant renal genetic and induced hypertension (28). Moreover, the ubiqui- dopaminergic system is important in the pathogenesis of some forms of genetic hypertension (3, 5, 7–17). Several mechanisms Abbreviations: GRK, G protein-coupled receptor kinase; CHO, Chinese hamster ovary; SNP, potentially responsible for the failure of endogenous renal single nucleotide polymorphism. dopamine to engender a natriuretic effect in genetic hyperten- †To whom reprint requests should be addressed at: c͞o Robin A. Felder, Department of sion have been investigated and ruled out, including decreased Pathology, Box 168, Charlottesville, VA 22908. E-mail: [email protected]. renal dopamine production and receptor expression, aberrant The publication costs of this article were defrayed in part by page charge payment. This nephron segment distribution of dopamine receptors, defective article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. effector (adenylyl cyclase or phospholipase C), and §1734 solely to indicate this fact.

3872–3877 ͉ PNAS ͉ March 19, 2002 ͉ vol. 99 ͉ no. 6 www.pnas.org͞cgi͞doi͞10.1073͞pnas.062694599 Downloaded by guest on September 28, 2021 Fig. 2. Phosphorylation of the D1 receptor in renal proximal tubule cells from Fig. 1. D1-like agonist stimulation of GRK activity in renal proximal tubule hypertensive (HT) subjects. Lysates of renal proximal tubule cells were immu- cells from hypertensive (HT) subjects. The D -like agonist fenoldopam (5 ␮M) 1 noprecipitated with a D1 receptor antibody and immunoblotted with an increased GRK activity (measured by the phosphorylation of rhodopsin) in HT anti-phosphoserine antibody. The amount of basal phosphorylated D1 recep- but not in normotensives (NT) with time. (Inset) Fenoldopam stimulation of tor was greater in hypertensive than in normotensive subjects. Fenoldopam (5 rhodopsin phosphorylation in HT (Ϸ40 kDa); addition of GRK4␥͞␦ antibody ␮M) increased the phosphorylation of the D1 receptor in cells from normo- (GRK4; Inset) decreased the 10-min phosphorylation of rhodopsin (Inset). GRK tensive but not in cells from hypertensive subjects. Sense or scrambled GRK4 activity was measured by the phosphorylation of rhodopsin (31). Of the four oligonucleotides had no significant effect on the quantity of phosphorylated isoforms of GRK4 in humans, only GRK4␣ can phosphorylate rhodopsin (25). D1 receptor in hypertensive or normotensive subjects. In contrast, antisense Because the D agonist did not stimulate GRK activity in cells from normoten- 1 GRK4 oligonucleotides inhibited the phosphorylation of the D1 receptor in sive subjects, the effect of GRK ␣͞␤ antibody was not tested. Number of both hypertensive or normotensive subjects (HT Ͼ NT). (Inset) Anti- studies: n ϭ 5͞group except at 1 min and 5 min where n ϭ 4͞group. #, P Ͻ 0.05 phosphoserine immunoblots of the anti-D1 receptor antibody immunopre- vs. 0 time, t test; *, P Ͻ 0.05 vs. 0 time, ANOVA for repeated measures, Scheffe´’s cipitates, unless otherwise indicated. Lanes 1–4, HT; lanes 6–9, NT; lanes 1 and test; a, P Ͻ 0.05 HT vs. NT, t test. Data are mean Ϯ SE. 6, basal phosphorylation; lanes 2 and 7, effect of fenoldopam; lanes 3 and 8, effect of GRK4 sense͞scrambled oligonucleotides; lanes 4 and 9, effect of GRK4 antisense oligonucleotides; lane 10, Western blot of human proximal tous expression of GRK2 and GRK3 is at odds with the recog- tubule cells with D1 antibody preadsorbed with the immunizing peptide; and nized preeminence of the kidney in the pathogenesis of both lane 11, Western blot of human proximal tubule cells with D1 antibody. Lane rodent and human essential hypertension (1, 2). The limited 5, molecular size marker, 80 kDa. The graph depicts the composite studies expression of GRK4 (24, 25) and the fact that the GRK4 gene from five hypertensive and four normotensive subjects. Homozygous GRK4 locus is linked to hypertension (29) make GRK4 an attractive gene variants were found in four of the five hypertensive subjects. The % area was normalized to 100% for either hypertensive or normotensive subjects. candidate for a pathogenetic mechanism in human hypertension. The inhibition of phosphorylation of the D1 receptor was associated with an Therefore, we sought to determine whether GRK4 is expressed enhancement of the fenoldopam-induced increase in cAMP accumulation in in renal proximal tubules and whether genetic variants of GRK4 hypertensive subjects (see Fig. 3). a, P Ͻ 0.05 normotensive vs. hypertensive, t affect renal function and blood pressure. test; *, P Ͻ 0.05 vs. other hypertensive groups, ANOVA, Scheffe´’s test; #, P Ͻ 0.05 vs. normotensive basal, ANOVA, Scheffe´’s test; and ϩ, P Ͻ 0.05 vs. Methods normotensive fenoldopam alone, t test. Data are mean Ϯ SE. Tissue Culture. Human kidneys were obtained as fresh surgical specimens from white patients who had unilateral nephrectomy because of renal carcinoma. The patient records of the subjects was used to create stable Chinese hamster ovary (CHO) cell line were reviewed and classified into those with either normal blood expressing the pTet-Off regulator plasmid (CLONTECH; ref. ␥ ␦ MEDICAL SCIENCES pressure (n ϭ 9) or essential hypertension (n ϭ 14). Subjects with 30). GRK4 and GRK4 cDNAs, obtained from reverse tran- systolic blood pressures less than 140 mm Hg and diastolic blood scription–PCR of mRNA from human kidney cortex, were pressures less than 90 mm Hg were considered normotensive. subcloned into a pTet-Off response plasmid (CLONTECH). Subjects with systolic blood pressures equal to or greater than Additionally, CHO cell lines stably transfected with rD1 were ␥ 140 mm Hg and͞or diastolic blood pressures equal to or greater transiently transfected with GRK4 wild type or variants with than 90 mm Hg and͞or on antihypertensive medications were TransIT-LT2 (Panvera, Madison, WI). considered hypertensive. Cultures of renal proximal tubule cells from histologically Determination of cAMP Accumulation. The cells were washed twice verified normal sections (5 ϫ 105 cells per well in 24-well plastic with D-PBS, after which 1 mM 3-isobutyl-1-methyl-xanthine was plates coated with 0.075% Type I collagen) were incubated at added to each well. The cells were incubated at 37°C for 30 min ͞ 37°C in 95% air 5% CO2 and grown in a serum-free medium in the presence or absence of drugs: D1-like receptor agonist, consisting of a 1:1 mixture of DMEM and Ham’s F-12 medium fenoldopam, or the D1-like receptor antagonist SCH23390 (Re- supplemented with selenium (5 ng͞ml), insulin (5 ␮g͞ml), search Biochemicals, Natick, MA), or forskolin (Sigma). Then, transferrin (5 ␮g͞ml), hydrocortisone (36 ng͞ml), triiodothyro- the cells were lysed with 0.1 M HCl and frozen at Ϫ80°C. cAMP nine (4 pg͞ml), and epidermal growth factor (10 ng͞ml) (16). concentration was measured by RIA (13, 16). Protein concen- When subconfluent (90–95%), the cells were subcultured (pas- tration was measured with the BCA protein assay kit (Pierce). sages 6–8) for use in experimental protocols by using trypsin- EDTA (0.05%, 0.02%). The culture conditions are conducive for Light Microscopic Immunohistochemistry. Immunohistochemistry growth of human renal proximal tubules that retain character- of kidney tissues and cells in culture fixed in HISTOCHOICE istics of renal proximal tubule cells (16). (Amresco, Solon, OH) was performed as described (16). Affin- ity-column purified polyclonal human D1 receptor antibodies Transfection and Cell Culture. The rat D1 (rD1) receptor cDNA was were raised against a synthetic peptide sequence GSGETQPFC subcloned in the expression vector pPUR (CLONTECH). Cal- (amino acids 299–307; ref. 16). Two commercially available cium phosphate-mediated transfection of the resulting construct GRK4 isoform antibodies were used (Santa Cruz Biotechnolo-

Felder et al. PNAS ͉ March 19, 2002 ͉ vol. 99 ͉ no. 6 ͉ 3873 Downloaded by guest on September 28, 2021 phosphorothioate oligonucleotides (5 nM) for 4–16 h. The cells were then washed and reincubated for a total incubation time of 24 to 72 h. Two antisense oligonucleotides were used: 5Ј-CAC GAT GTT CTC GAG CTC CAT-3Ј, complementary to bases 255–275, and 5Ј-CTC CAT GTC CTG GCG CCG-3Ј, comple- mentary to bases 243–260 (25). Sense (5Ј-ATG GAG CTC GAG AAC ATC GTG-3Ј) or scrambled antisense (5Ј-ACC CTT GCG TCC GCT GCG-3Ј) oligonucleotides were used as controls. These sequences are common to all GRK4 isoforms. GRK4 and GRK6 have the same sequence in the first 18 bases but not in the 5Ј non-coding region (24, 25, 32).

Immunoprecipitation. Proximal tubule cells were incubated with vehicle, fenoldopam, sense, scrambled, or antisense propyne͞ phosphorothioate GRK4 oligonucleotides (5 nM) as described above. The membranes were lysed with ice-cold lysis buffer (PBS͞1% Nonidet P-40͞0.5% sodium deoxycholate͞0.1% Fig. 3. Normalization of D1-like agonist-induced stimulation of cAMP accu- SDS͞1 mM EDTA͞1 mM EGTA͞1 mM sodium vanadate͞1mM mulation in renal proximal tubule cells from hypertensive (HT) subjects by NaF͞1 mM PMSF͞10 ␮g/ml aprotinin͞10 ␮g/ml leupeptin). The GRK4 antisense oligonucleotides. GRK4 antisense propyne͞phosphorothioate oligonucleotides normalized the ability of fenoldopam to stimulate cAMP lysates were incubated with IgG-purified anti D1 receptor on ice for 1 h and protein-A agarose for 12 h with rocking at 4°C. The accumulation in HT; a slight increase was noted in normotensives (NT). GRK4 ͞ sense or scrambled oligonucleotides did not significantly affect the ability of proteins separated by SDS PAGE were electrophoretically fenoldopam to stimulate cAMP accumulation in either NT or HT. Basal cAMP transferred onto nitrocellulose membranes. The transblot sheets production was similar in hypertensive (940 Ϯ 34 fmol͞mg protein͞30 min, n ϭ were blocked with 5–10% nonfat dry milk in 10 mM Tris⅐HCl 8) and normotensive subjects (1,015 Ϯ 36 fmol͞mg protein͞30 min, n ϭ 6; P ϭ (pH 7.5), 150 mM NaCl, and 0.1% Tween-20 and were incubated 0.10, t test). GRK4␥͞␦ immmunoreactive levels (Inset) and GRK4␣͞␤ (not with diluted affinity-purified polyclonal anti-phosphoserine an- shown) were attenuated by antisense (5Ј-CAC GAT GTT CTC GAG CTC CAT-3Ј; tibody (Zymed; ref. 16). In some cases, the cells were labeled ͞ lane 2, NT; lane 5, HT) but not by sense scrambled oligonucleotides (lane 3, NT; with 32P and immunoprecipitated with anti-D receptor anti- lane 6, HT) compared with vehicle-treated controls (lane 1, NT; lane 4, HT). 1 Results were similar by using two different oligonucleotides as described in body. The autoradiograms and immunoblots, visualized with the Methods. Four of the eight hypertensive subjects were homozygous in at least ECL system (Amersham Pharmacia), were quantified by one of the polymorphic sites (nucleotide positions 448 and 679, n ϭ 1; 679, n ϭ densitometry (16). 1; and 1711, n ϭ 2). Uncoupling without GRK4 gene variants could be inter- preted to indicate the presence of other meaningful GRK4 polymorphisms or Transgenic Mice. Two constructs were used to generate transgenic there other causes of uncoupling besides GRK4 gene variants. Data are mice. The full-length wild-type hGRK4␥ cDNA was obtained by mean Ϯ SE. Number of experiments is in parentheses. Each n represents cells PCR by using the GRK4␣ cDNA in pTRE plasmid as template. from one subject. a, P Ͻ 0.05 NT vs. HT, t test; *, P Ͻ 0.05 vs. other groups in The A142V polymorphism was obtained by using site-directed HT, ANOVA for repeated measures, Scheffe´’s test. mutagenesis. The two cDNAs were subcloned into pcDNA3.1. Expression of the cDNA insert was under the control of the gy); one GRK4 antibody recognized both the ␣ and ␤ isoforms, cytomegalovirus promoter and bovine growth hormone (BGH) poly(A) signal. Full-length cDNA was verified by sequencing. whereas another recognized both the ␥ and ␦ isoforms. The The mice were produced by microinjecting the cDNA con- specificity of these antibodies has been previously reported (24). structs into fertilized eggs obtained from the mating of a (C57BL͞6J ϫ SJL͞J) F female mouse and a (C57BL͞6J ϫ Determination of GRK Activity. GRK activity was measured ac- 1 SJL͞J) F male mouse at the University of Michigan Transgenic cording to Benovic (31). Renal proximal tubular extracts were 1 Animal Model Core. The presence of the transgene in the prepared by homogenization in ice-cold lysis buffer containing ⅐ transgenic mice was verified by PCR. The first set of PCR (in mM): 25 Tris HCl (pH 7.5), 5 EDTA, 5 EGTA with leupeptin primers (pcDNA, sense 5Ј-CGACTCACTATAGGGAGAC-3Ј; (10 ␮g͞ml), aprotinin (20 ␮g͞ml), and 1 PMSF. The crude Ј Ј ϫ hGRK4, antisense 5 -ATGGTTCCCCTCTTAGGTAG-3 ) gen- homogenate was centrifuged at 30,000 g for 30 min. The pellet erated a 530-bp fragment. The second set PCR primers (pcDNA, was solubilized by 200 mM NaCl on ice for 30 min and Ј Ј ϫ sense 5 -CGACTCACTATAGGGAGAC-3 ; hGRK4, antisense centrifuged at 30,000 g for 30 min. The supernatant was used 5Ј-CTTGATTCTTTGATCGACCTCCTCCC-3Ј) generated a for all GRK assays and immunoblotting. Twenty micrograms of 1,260-bp fragment. Twenty-two mice carrying wild-type protein extract was incubated with rhodopsin-enriched rod outer hGRK4␥ and 10 mice carrying hGRK4␥ A142V were identified. segments in assay buffer with 10 mM MgCl2 and 0.1 mM ATP Both wild-type and A142V hGRK4␥ were expressed in the ␥ 32 (containing [ - P]ATP). After incubation in white light for 15 kidney as determined by reverse transcription–PCR, immuno- min at room temperature, the reaction was stopped with ice-cold blotting, and immunohistochemistry. lysis buffer and centrifuged at 13,000 ϫ g for 15 min. The pellet was resuspended in Laemmli buffer and subjected to 12% Blood Pressure and Renal Functional Studies. The animal studies SDS͞PAGE. The gels were subjected to autoradiography, and were approved by the Georgetown University Animal Care and the phosphorylated rhodopsin was quantified by using both Use Committee. The mice were anesthetized with pentobarbital densitometry and radioactive counting of the excised bands at (50 mg͞kg i.p.), placed on a heated board to maintain body the appropriate size. GRK activity was also measured in the temperature at 37°C, and tracheotomized (33). Catheters were presence or absence of a GRK4 ␥͞␦ antibody. inserted into the femoral vessels for fluid administration, blood drawing, and blood pressure monitoring. Urine was collected via GRK4 Oligonucleotides. The effects of inhibition of GRK4 activity a suprapubic cystostomy. After a 60-min equilibration period, a on cAMP accumulation and levels of immunoreactive GRK4 ␥͞␦ baseline 30-min collection period was obtained. Thereafter, were determined by treating renal proximal tubule cells with fenoldopam was infused intravenously at 2 ␮g͞kg body weight͞ vehicle, sense, scrambled, or antisense GRK4 propyne͞ min for 30 min. Urine was collected during the drug infusion and

3874 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.062694599 Felder et al. Downloaded by guest on September 28, 2021 sion of the experiment, the mice were killed with an i.v. injection of pentobarbital (100 mg͞kg).

Statistical Analysis. The data are expressed as means Ϯ SE. Significant differences among and within groups were deter- mined by ANOVA for n Ͼ 2 and t test for n ϭ 2. N refers to the number of treatment groups or experiments, cell lines, or mice as indicated. Results and Discussion GRK4 was previously thought to be expressed mainly in testes and the brain (24, 25). However, GRK4␥ has been reported to be expressed in human myometrium (34). We detected mRNA of all of the reported GRK4 isoforms in renal proximal tubules (not shown). There were no differences in the protein expression of the isoforms of GRK4 (␣͞␤, ␥͞␦) in kidneys or cultured renal proximal tubule cells between hypertensive and normotensive subjects (not shown). However, basal and D1 agonist-stimulated GRK activities were elevated in renal proximal tubular cells from hypertensive subjects (Fig. 1). The increased GRK activity in cells from hypertensive subjects was probably caused, in part, by GRK4␥͞␦ because the antibody that recognizes these two GRK4 isoforms blocked the stimulatory effect of the D1-like agonist, fenoldopam, on GRK activity. An initial step in the desensitization process is the phosphor- ylation of the G protein-coupled receptor by GRK (18, 19). In our studies, basal (ligand-independent) GRK activity and serine phosphorylation of the D1 receptor in renal proximal tubule cells was higher in hypertensive subjects than in normotensive sub- jects (Figs. 1 and 2). As expected, fenoldopam increased the phosphorylation of the D1 receptor and cAMP accumulation in cells from normotensive subjects. In contrast, fenoldopam failed Fig. 4. (A) Effect of GRK4␥ variants on D1-like agonist stimulation of cAMP to increase further the phosphorylation of the D1 receptor and production in CHO cells stably transfected with D1 receptor and GRK4␥ in tet-off vector. The filled symbols represent CHO cells treated with tetracycline in which minimally increased cAMP accumulation in cells from hyper- GRK4␥ was turned off and served, therefore, as controls. In the absence of GRK4␥ tensive subjects (Figs. 2 and 3). The increased GRK4 activity was Ϫ5 expression, fenoldopam (10 M) increased cAMP accumulation to a similar related to the diminished responses of the D1 receptor in extent in wild-type (W) and single (R65L, A142V,orA486V) or double variant hypertension because antisense GRK4 oligonucleotides com- (R65L and A486V)-transfected cells. The open symbols represent CHO cells not pletely blocked the serine phosphorylation of the D1 receptor treated with tetracycline and therefore, GRK4␥ was expressed. Expression of and restored the ability of fenoldopam to stimulate cAMP ␥ E wild-type GRK4 (W, ) decreased the ability of fenoldopam to stimulate cAMP accumulation in cells from hypertensive subjects. In contrast, in accumulation. The decrease was greater with GRK4␥ variants at R65L (CGT to CTT; ƒ), or A486V (GCG to GTG; ᮀ) and even greater with A142V (GCC to GTC; ‚) and cells from normotensive subjects, antisense GRK4 slightly in- the double variant (R65L and A486V; ࡗ). Studies were performed in CHO cells creased the ability of fenoldopam to stimulate cAMP accumu- expressing similar amounts of GRK4␥ and D1 receptor. Fenoldopam had no effect lation and also diminished the magnitude of phosphorylation of

on cAMP accumulation in untransfected CHO cells, CHO cells with tet-off regu- the D1 receptor (Figs. 2 and 3). Antisense GRK4 oligonucleo- MEDICAL SCIENCES lator, or response plasmid alone, or CHO cells expressing only GRK4␥ without the tides did not affect basal or forskolin-stimulated cAMP produc- D1 receptor (data not shown). Basal cAMP accumulation was similar among the tion (data not shown). Compared with fenoldopam alone, nei- groups in the presence or absence of tetracycline (527 Ϯ 5 fmol͞mg protein with ther sense nor scrambled GRK4 oligonucleotides affected cAMP tetracycline͞30 min and 522 Ϯ 5 fmol͞mg protein without tetracycline͞30 min). accumulation or receptor serine phosphorylation in either Ϯ Data are mean SE (error bars are absent if the symbols are bigger than the error group. The almost complete suppression of the phosphorylation bars). n, The number of cell lines studied per group, performed in triplicate. #, P Ͻ of the D1 receptor and normalization of the cAMP response by 0.05 wild-type vs. R65L ϩ A486V, A142V; *, P Ͻ 0.05 wild-type vs. others, ANOVA, Scheffe´’s test (cells not treated with tetracycline). (B) Effect of GRK4␥ variants on GRK4 antisense oligonucleotides in renal proximal tubules from serine phosphorylation of the D1 receptor in CHO cells stably (n ϭ 3) or transiently hypertensive subjects suggest that the major GRK involved in the (n ϭ 5) transfected with rD1 receptor and GRK4␥ in tet-off vector. Lysates of CHO phosphorylation and desensitization of the D1 receptor in hy- cells were immunoprecipitated with anti-D1 antibody and immunoblotted with pertension is GRK4 rather than other GRKs that may be an anti-phosphoserine antibody (in two studies, the cells were labeled with 32P). expressed in this nephron segment. (Inset) Compared with untransfected CHO cells (lane 1) and CHO cells transfected Several single nucleotide polymorphisms (SNPs) have been ␥ with wild-type GRK4 (lane 2), phosphorylation of the D1 receptor was increased noted in the coding region of the GRK4 gene, but their clinical ␥ in CHO cells transfected with the GRK4 variants R65L (lane 3), A486V (lane 4), relevance has not been investigated (24). However, the GRK4 R65L͞A486V (lane 5), and A142V (lane 6). *, P Ͻ 0.05 vs. GRK4␥ wild-type or nontransfected CHO cell; #, P Ͻ 0.05 vs. A142V or R65L͞A486V, ANOVA, locus is linked with essential hypertension (29). Sequencing of Duncan’s test. GRK4 cDNAs from normotensive and hypertensive subjects confirmed the presence of SNPs (24): nucleotide 448, CGT to CTT (amino acid R65L) and the two reported variants, nucle- for another two 30-min periods, thereafter. Blood (50 ␮l) was otide 679, GCC to GTC (amino acid A142V), and nucleotide obtained from the femoral artery before the drug infusion and 1711, GCG to GTG (amino acid A486V). at the end of the last urine collection period. In some mice, bolus The increased GRK activity in cells from hypertensive subjects injections of fenoldopam (1, 10, 100, and 1000 ng) and the effect was attenuated by antibodies to GRK4␥͞␦; therefore, we deter- on blood pressure were monitored for 10 min, and the time for mined whether the variations in the GRK4␥ or GRK4␦ blood pressure to recover to preinjection levels. At the conclu- have any functional consequences. Because we did not find any

Felder et al. PNAS ͉ March 19, 2002 ͉ vol. 99 ͉ no. 6 ͉ 3875 Downloaded by guest on September 28, 2021 Table 1. Baseline characteristics of hGRK4␥ transgenes in anesthetized mice Variables Wild type hGRK4␥ (n ϭ 22) A142V hGRK4␥ (n ϭ 7)

Body weight, g 26 Ϯ 128Ϯ 2 Kidney weight, % body weight 1.54 Ϯ 0.04 1.51 Ϯ 0.07 Heart weight, % body weight 0.46 Ϯ 0.01 0.52 Ϯ 0.02* Heart rate, beats͞min 396 Ϯ 15 431 Ϯ 16 Urine flow, ␮l͞min 1.38 Ϯ 0.42 1.63 Ϯ 0.50 Sodium excretion, nEq͞min 222 Ϯ 90 209 Ϯ 103 Systolic blood pressure, mm Hg 97 Ϯ 2 129 Ϯ 2* Diastolic blood pressure, mm Hg 68 Ϯ 297Ϯ 4* Mean blood pressure, mm Hg 77 Ϯ 2 108 Ϯ 3*

Data are mean Ϯ SE. *, P Ͻ 0.05 vs. wild-type transgene. The mice were 3–4 mo of age.

␦ effect of GRK4 on D1 receptor function (unpublished obser- to a greater extent in salt-sensitive hypertensive than in normo- vations), we concentrated our studies on GRK4␥. The ability of tensive subjects (15). The ability of dopamine to relax renal fenoldopam to stimulate cAMP accumulation in CHO cells (in artery strips is also increased in stroke-prone SHRs (42). How- the absence of GRK4␥ expression) was similar to that noted in ever, fenoldopam has been reported to fail to vasodilate the HEK-293 cells, a cell with low endogenous GRK activity (Fig. kidney of some patients with essential hypertension (43). The 4A; ref. 35). The expression of wild-type GRK4␥ slightly de- ability of fenoldopam to counteract the renal vasoconstrictor creased the ability of the D1 agonist to stimulate cAMP pro- effect of exogenous angiotensin II and inhibit tubuloglomerular duction (Fig. 4A). However, the D1 receptor-mediated cAMP feedback is also impaired in the SHR (44, 45). It is possible that production was markedly impaired by GRK4␥ SNPs (R65L, differences in published reports may be related to differences in A142V, A486V, and combined R65L plus A486V). The effect of classes of hypertension being studied (salt-sensitive vs. salt- wild-type or GRK4␥ SNPs was not due to differences in the resistant) or different experimental conditions (basal tone vs. ␥ quantity of the expression of either the D1 receptor or GRK4 (not shown). Wild-type GRK4␥ or its variants did not affect the ability of forskolin to stimulate cAMP accumulation (not shown), indicating specificity of the interaction of GRK4␥ with the D1 receptor. The action of fenoldopam was selective for the D1 receptor because the fenoldopam effect was blocked by the D1-like antagonist SCH23390 (not shown). Expression of GRK4␥ SNPs was also associated with increased basal phos- phorylation of the D1 receptor (Fig. 4B). These studies suggest that the increased basal phosphorylation of the D1 receptor by GRK4␥, may explain, in part, the decreased responsiveness of ␣ ␤ ␥ the D1 receptor in hypertension. Of the GRK4 isoforms ( , , , and ␦), only GRK4␣ and GRK4␦ have been reported to be involved in the regulation of G protein-coupled receptors (e.g., ␤-adrenergic, luteinizing hormone, metabotropic glutamate, and M2 muscarinic receptors; refs. 24 and 35–40). GRK4␥ may preferentially regulate D1 receptors because it has not been found to have much activity in regulating other G protein- coupled receptors (24). Of the three GRK4␥ SNPs, GRK4␥ A142V had the most drastic effect on D1 receptor function in the expression studies. There- fore, we compared the consequences of the expression of GRK4␥ wild type and GRK4␥ A142V in transgenic mice. The important role that GRK4␥ SNPs play in the regulation of blood pressure was buttressed by the demonstration that transgenic mice ex- pressing GRK4␥ A142V were hypertensive whereas those ex- pressing the wild-type GRK4␥ were normotensive (Table 1). Furthermore, the heart weights were greater in hypertensive GRK4␥ A142V than in normotensive wild-type GRK4␥ mice. The hypotensive effects of bolus i.v. administration of fenoldopam ␥ Fig. 5. Effect of the D1-like agonist, fenoldopam, in anesthetized mice were comparable in wild-type and GRK4 A142V transgenes overexpressing wild-type (n ϭ 10) or A142V (n ϭ 4) GRK4␥.(A) Effect of bolus (Fig. 5A). However, the i.v. infusion of fenoldopam increased i.v. injections of fenoldopam (1–1000 ␮g) on arterial blood pressure was urine flow and sodium excretion in GRK4␥ wild-type mice but observed for 10 min. (B) Effect of a 30-min i.v. infusion of fenoldopam (2 not in the hypertensive GRK4␥ A142V transgenes (Fig. 5B). ␮g͞kg͞min) on sodium excretion (UNaV) and urine flow (V). Percentage These results are in agreement with previous studies showing changes from a 30-min vehicle infusion period are depicted. *, P Ͻ 0.05 vs. basal, ANOVA on ranks, Dunnet’s test; ϩ, P Ͻ 0.05 vs. basal, t test; #, P Ͻ 0.05 that the desensitization of the D1 receptor in genetic hyperten- GRK4␥ A142V vs. WT (wild-type). Blood pressures were not affected by sion is renal specific (3, 7, 8, 16). The renal vasodilatory effect fenoldopam infusion except for transient decreases in GRK4␥ A142V to the of D1-like agonists may be preserved in genetic hypertension (3). same level as the wild-type transgenes during recovery 1 (not shown). Differ- Thus, the ability of fenoldopam to decrease renal vascular ences in blood pressure and renal responses to D1-like agonist infusion cannot resistance is maintained in the spontaneously hypertensive rat be explained by differences in GRK4␥ or D1 receptor expression (data not (SHR) (41). Fenoldopam increases effective renal plasma flow shown).

3876 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.062694599 Felder et al. Downloaded by guest on September 28, 2021 reactivity to vasoconstrictors; ref. 3). Nevertheless, the preser- development of hypertension (1, 2). The desensitization of the vation of the extrarenal vasodilatory, and the ‘‘distal’’ renal D1 receptor in renal proximal tubules in hypertension may lead tubular responses to D1-like agonists explains the ability of to a decreased ability of the kidney to eliminate a sodium fenoldopam, a D1-like agonist, to decrease blood pressure and chloride load (3, 6, 13, 15). Genes that regulate renal sodium produce a natriuresis in hypertensive subjects (15, 46). transport are important in the regulation of blood pressure (50). Genes regulating the renin angiotensin system have been In summary, we have found a ligand-independent increase in implicated in the pathogenesis of essential hypertension (47, 48). GRK activity and decrease in D1 receptor function in renal In a cohort of Ghanian hypertensive subjects, we found that proximal tubule cells in human essential hypertension because of there was a significant interaction of GRK4 SNPs (termed FJ) activating SNPs of GRK4␥. These studies show a candidate gene, with the SNPs of angiotensin converting enzyme, AT1 angio- whose locus is linked to (29), and whose SNPs are associated tensin receptor, and angiotensinogen (49). In a white population, with, essential hypertension, which has been shown to have hypertensives (n ϭ 89) and normotensives (n ϭ 90) differed pathophysiologic consequences in renal proximal tubule and significantly in disease risk as a function of R65L͞A486V hap- CHO cells, to impair renal sodium excretion, and to cause lotypes (unpublished data). Similar effects were also found in a hypertension in transgenic mice. We describe here a transgenic Japanese population (hypertensives, n ϭ 122, normotensives, n mouse model in which the pathogenesis of hypertension appears ϭ 54), where normal subjects were deficient in 65L͞486V alleles, to mimic that reported for human essential hypertension. This and interactions among A142V, A486V, and angiotensin con- model may be useful in the study of new approaches to the verting enzyme alleles significantly associated with disease phe- treatment of hypertension. notypes (unpublished data). These GRK4 SNPs impaired the function of D receptors, whether endogenously (renal proximal We wish to acknowledge Dr. Jeffrey L. Benovic for initially providing the 1 rhodopsin used in the GRK assay as well as the monoclonal GRK2 tubule cells) or exogenously (CHO cells) expressed, and in- antibody. These studies were supported in part by grants from the creased blood pressure and impaired the diuretic and natriuretic National Institutes of Health, the National Institute of Diabetes and effects of D1-like agonist stimulation. The failure of the kidney Digestive and Kidney Diseases, the National Heart, Lung, and Blood to excrete excess sodium chloride is thought to be crucial in the Institute, and The National Center for Research Resources.

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