G protein-coupled receptor kinase 4 gene variants in human essential hypertension

Abstract

Essential hypertension has a heritability as high as 30-50%, but its genetic cause(s) has not been determined despite intensive investigation. The renal dopaminergic system exerts a pivotal role in maintaining fluid and electrolyte balance and participates in the pathogenesis of genetic hypertension. In genetic hypertension, the ability of dopamine and D(1)-like agonists to increase urinary sodium excretion is impaired. A defective coupling between the D(1) dopamine receptor and the G protein/effector enzyme complex in the proximal tubule of the kidney is the cause of the impaired renal dopaminergic action in genetic rodent and human essential hypertension. We now report that, in human essential hypertension, single nucleotide polymorphisms of a G protein-coupled receptor kinase, GRK4gamma, increase G protein-coupled receptor kinase (GRK) activity and cause the serine phosphorylation and uncoupling of the D(1) receptor from its G protein/effector enzyme complex in the renal proximal tubule and in transfected Chinese hamster ovary cells. Moreover, expressing GRK4gammaA142V but not the wild-type gene in transgenic mice produces hypertension and impairs the diuretic and natriuretic but not the hypotensive effects of D(1)-like agonist stimulation. These findings provide a mechanism for the D(1) receptor coupling defect in the kidney and may explain the inability of the kidney to properly excrete sodium in genetic hypertension.

Figure 1

D₁-like agonist stimulation of GRK activity in renal proximal tubule cells from hypertensive (HT) subjects. The D₁-like agonist fenoldopam (5 μM) increased GRK activity (measured by the phosphorylation of rhodopsin) in HT but not in normotensives (NT) with time. (Inset) Fenoldopam stimulation of rhodopsin phosphorylation in HT (≈40 kDa); addition of GRK4γ/δ antibody (GRK4; Inset) decreased the 10-min phosphorylation of rhodopsin (Inset). GRK activity was measured by the phosphorylation of rhodopsin (31). Of the four isoforms of GRK4 in humans, only GRK4α can phosphorylate rhodopsin (25). Because the D₁ agonist did not stimulate GRK activity in cells from normotensive subjects, the effect of GRK α/β antibody was not tested. Number of studies: n = 5/group except at 1 min and 5 min where n = 4/group. #, P < 0.05 vs. 0 time, t test; *, P < 0.05 vs. 0 time, ANOVA for repeated measures, Scheffé's test; a, P < 0.05 HT vs. NT, t test. Data are mean ± SE.

Figure 2

Phosphorylation of the D₁ receptor in renal proximal tubule cells from hypertensive (HT) subjects. Lysates of renal proximal tubule cells were immunoprecipitated with a D₁ receptor antibody and immunoblotted with an anti-phosphoserine antibody. The amount of basal phosphorylated D₁ receptor was greater in hypertensive than in normotensive subjects. Fenoldopam (5 μM) increased the phosphorylation of the D₁ receptor in cells from normotensive but not in cells from hypertensive subjects. Sense or scrambled GRK4 oligonucleotides had no significant effect on the quantity of phosphorylated D₁ receptor in hypertensive or normotensive subjects. In contrast, antisense GRK4 oligonucleotides inhibited the phosphorylation of the D₁ receptor in both hypertensive or normotensive subjects (HT > NT). (Inset) Anti-phosphoserine immunoblots of the anti-D₁ receptor antibody immunoprecipitates, unless otherwise indicated. Lanes 1–4, HT; lanes 6–9, NT; lanes 1 and 6, basal phosphorylation; lanes 2 and 7, effect of fenoldopam; lanes 3 and 8, effect of GRK4 sense/scrambled oligonucleotides; lanes 4 and 9, effect of GRK4 antisense oligonucleotides; lane 10, Western blot of human proximal tubule cells with D₁ antibody preadsorbed with the immunizing peptide; and lane 11, Western blot of human proximal tubule cells with D₁ antibody. Lane 5, molecular size marker, 80 kDa. The graph depicts the composite studies from five hypertensive and four normotensive subjects. Homozygous GRK4 gene variants were found in four of the five hypertensive subjects. The % area was normalized to 100% for either hypertensive or normotensive subjects. The inhibition of phosphorylation of the D₁ receptor was associated with an enhancement of the fenoldopam-induced increase in cAMP accumulation in hypertensive subjects (see Fig. 3). a, P < 0.05 normotensive vs. hypertensive, t test; *, P < 0.05 vs. other hypertensive groups, ANOVA, Scheffé's test; #, P < 0.05 vs. normotensive basal, ANOVA, Scheffé's test; and +, P < 0.05 vs. normotensive fenoldopam alone, t test. Data are mean ± SE.

Figure 3

Normalization of D₁-like agonist-induced stimulation of cAMP accumulation in renal proximal tubule cells from hypertensive (HT) subjects by GRK4 antisense oligonucleotides. GRK4 antisense propyne/phosphorothioate oligonucleotides normalized the ability of fenoldopam to stimulate cAMP accumulation in HT; a slight increase was noted in normotensives (NT). GRK4 sense or scrambled oligonucleotides did not significantly affect the ability of fenoldopam to stimulate cAMP accumulation in either NT or HT. Basal cAMP production was similar in hypertensive (940 ± 34 fmol/mg protein/30 min, n = 8) and normotensive subjects (1,015 ± 36 fmol/mg protein/30 min, n = 6; P = 0.10, t test). GRK4γ/δ immmunoreactive levels (Inset) and GRK4α/β (not shown) were attenuated by antisense (5′-CAC GAT GTT CTC GAG CTC CAT-3′; lane 2, NT; lane 5, HT) but not by sense/scrambled oligonucleotides (lane 3, NT; lane 6, HT) compared with vehicle-treated controls (lane 1, NT; lane 4, HT). Results were similar by using two different oligonucleotides as described in Methods. Four of the eight hypertensive subjects were homozygous in at least one of the polymorphic sites (nucleotide positions 448 and 679, n = 1; 679, n = 1; and 1711, n = 2). Uncoupling without GRK4 gene variants could be interpreted to indicate the presence of other meaningful GRK4 polymorphisms or there other causes of uncoupling besides GRK4 gene variants. Data are mean ± SE. Number of experiments is in parentheses. Each n represents cells from one subject. a, P < 0.05 NT vs. HT, t test; *, P < 0.05 vs. other groups in HT, ANOVA for repeated measures, Scheffé's test.

Figure 4

(A) Effect of GRK4γ variants on D₁-like agonist stimulation of cAMP production in CHO cells stably transfected with D₁ receptor and GRK4γ in tet-off vector. The filled symbols represent CHO cells treated with tetracycline in which GRK4γ was turned off and served, therefore, as controls. In the absence of GRK4γ expression, fenoldopam (10⁻⁵ M) increased cAMP accumulation to a similar extent in wild-type (W) and single (R65L, A142V, or A486V) or double variant (R65L and A486V)-transfected cells. The open symbols represent CHO cells not treated with tetracycline and therefore, GRK4γ was expressed. Expression of wild-type GRK4γ (W, ○) decreased the ability of fenoldopam to stimulate cAMP accumulation. The decrease was greater with GRK4γ variants at R65L (CGT to CTT; ▿), or A486V (GCG to GTG; □) and even greater with A142V (GCC to GTC; ▵) and the double variant (R65L and A486V; ♦). Studies were performed in CHO cells expressing similar amounts of GRK4γ and D₁ receptor. Fenoldopam had no effect on cAMP accumulation in untransfected CHO cells, CHO cells with tet-off regulator, or response plasmid alone, or CHO cells expressing only GRK4γ without the D₁ receptor (data not shown). Basal cAMP accumulation was similar among the groups in the presence or absence of tetracycline (527 ± 5 fmol/mg protein with tetracycline/30 min and 522 ± 5 fmol/mg protein without tetracycline/30 min). Data are mean ± SE (error bars are absent if the symbols are bigger than the error bars). n, The number of cell lines studied per group, performed in triplicate. #, P < 0.05 wild-type vs. R65L + A486V, A142V; *, P < 0.05 wild-type vs. others, ANOVA, Scheffé's test (cells not treated with tetracycline). (B) Effect of GRK4γ variants on serine phosphorylation of the D₁ receptor in CHO cells stably (n = 3) or transiently (n = 5) transfected with rD₁ receptor and GRK4γ in tet-off vector. Lysates of CHO cells were immunoprecipitated with anti-D₁ antibody and immunoblotted with an anti-phosphoserine antibody (in two studies, the cells were labeled with ³²P). (Inset) Compared with untransfected CHO cells (lane 1) and CHO cells transfected with wild-type GRK4γ (lane 2), phosphorylation of the D₁ receptor was increased in CHO cells transfected with the GRK4γ variants R65L (lane 3), A486V (lane 4), R65L/A486V (lane 5), and A142V (lane 6). *, P < 0.05 vs. GRK4γ wild-type or nontransfected CHO cell; #, P < 0.05 vs. A142V or R65L/A486V, ANOVA, Duncan's test.

Figure 5

Effect of the D₁-like agonist, fenoldopam, in anesthetized mice overexpressing wild-type (n = 10) or A142V (n = 4) GRK4γ. (A) Effect of bolus i.v. injections of fenoldopam (1–1000 μg) on arterial blood pressure was observed for 10 min. (B) Effect of a 30-min i.v. infusion of fenoldopam (2 μg/kg/min) on sodium excretion (UNaV) and urine flow (V). Percentage changes from a 30-min vehicle infusion period are depicted. *, P < 0.05 vs. basal, ANOVA on ranks, Dunnet's test; +, P < 0.05 vs. basal, t test; #, P < 0.05 GRK4γ A142V vs. WT (wild-type). Blood pressures were not affected by fenoldopam infusion except for transient decreases in GRK4γ A142V to the same level as the wild-type transgenes during recovery 1 (not shown). Differences in blood pressure and renal responses to D₁-like agonist infusion cannot be explained by differences in GRK4γ or D₁ receptor expression (data not shown).