G protein-coupled receptor kinase 4 gene 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as well as in rodents with genetic hypertension maintaining fluid and electrolyte balance and participates in the (unpublished studies).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 dopamine 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 enzyme 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 kinases (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 rhodopsin kinase ␤ 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 enzymes (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;