Angiotensin IV induces tyrosine phosphorylation of focal adhesion kinase and paxillin in proximal tubule cells

Jian-Kang Chen1, Joe Zimpelmann1, Raymond C. Harris2, and Kevin D. Burns1

1 Division of Nephrology, Department of Medicine, Kidney Research Center, Ottawa Hospital Research Institute, University of Ottawa, Ottawa, Ontario, Canada K1H 8L6; and 2 Department of Medicine, Vanderbilt University, Nashville, Tennessee 37232


    ABSTRACT
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Angiotensin IV (ANG IV), the COOH-terminal hexapeptide fragment of angiotensin II (ANG II), binds to specific sites in the kidney, distinct from type 1 (AT1) and type 2 (AT2) receptors and designated type 4 (AT4) receptors. We determined signaling pathways for ANG IV in a proximal tubular cell line, LLC-PK1/Cl4. In these cells, we found no specific binding of [125I]-ANG II. In contrast, ANG IV dose dependently competed for [125I]-labeled ANG IV binding, with no displacement by either ANG II, the AT1 receptor antagonist losartan, or the AT2 antagonist PD-123319. Saturation binding indicated the presence of AT4 receptors of high affinity [dissociation constant (Kd) = 1.4 nM]. ANG IV did not affect cAMP or cGMP production and did not increase cytosolic calcium concentration in these cells. In contrast, immunoprecipitation and immunoblotting studies revealed that ANG IV caused dose-dependent tyrosine phosphorylation of p125-focal adhesion kinase (p125-FAK) and p68-paxillin within 2 min, with maximal stimulation at 30 min. ANG IV-stimulated tyrosine phosphorylation of p125-FAK and paxillin was not affected by pretreatment with either losartan or PD-123319, and ANG II (10-7 M) did not induce protein tyrosine phosphorylation. Our results indicate that LLC-PK1/Cl4 cells express ANG IV receptors, which we demonstrate for the first time are linked to tyrosine phosphorylation of focal adhesion-associated proteins. This suggests that ANG IV, a product of ANG II metabolism, may regulate function of the focal adhesion complex in proximal tubule cells.

renin-angiotensin system; receptor; signaling; tyrosine kinase; renal proximal tubular epithelial cells


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THE CLASSIC INTRARENAL FUNCTIONS of angiotensin II (ANG II) are primarily mediated by binding to G protein-coupled type 1 (AT1) receptors (43, 46), whereas the type 2 (AT2) receptors may be involved in natriuresis, growth inhibition, and apoptosis (19, 30, 50). In contrast, much less attention has been paid to the COOH-terminal 3-8 hexapeptide (Val-Tyr-Ile-His-Pro-Phe) fragment of ANG II, known as ANG IV, which is generated from successive cleavage of ANG II by aminopeptidases A and N, and potentially from ANG I-(3-10) by an angiotensin-converting enzyme (ACE)-dependent pathway (21, 48). Until 1992, ANG IV was believed to be an inactive metabolic fragment of ANG II, but in that year it was reported to possess biological activity in the central nervous system by interacting with distinct binding sites (29, 31, 42).

With the use of autoradiography and radioligand binding studies, specific receptors for ANG IV, termed AT4 receptors (29, 31, 42), have been detected in cerebral cortex, hippocampus, basal ganglia, cerebellum, and spinal cord, as well as in several peripheral tissues including kidney, bladder, heart, spleen, prostate, adrenal, and colon (48, 49). The function of AT4 receptors remains incompletely understood, although several lines of evidence suggest that ANG IV is an important regulator of blood flow. When infused into the renal artery, ANG IV increased endothelium-dependent renal cortical blood flow without altering systemic blood pressure (14, 42). Similarly, when added to pulmonary arterial rings, ANG IV caused endothelium-dependent vasorelaxation, which was linked to activation of nitric oxide (NO) synthase and generation of cGMP by pulmonary arterial endothelial cells (34), and when topically applied in the brain, ANG IV potentiated L-arginine-dependent vasodilation of rabbit cerebral arterioles (24). In contrast, ANG IV has also been reported to cause renal, mesenteric, and pulmonary vasoconstrictor effects that were inhibited by the AT1 receptor antagonist losartan (13, 20), suggesting that ANG IV is a weak agonist of AT1 receptors.

ANG IV may also regulate cell growth responses. ANG IV promotes cell growth in rabbit cardiac fibroblasts (47), bovine coronary endothelial cells (25), and rat anterior pituitary cells (35). Interestingly, Kakinuma et al. (32) reported an antiapoptotic role for ANG IV in neuronal cells, but the mechanism for this effect remains unclear.

The kidney expresses abundant AT4 receptors, localized to the proximal convoluted and straight tubules in the cortex and outer stripe of the outer medulla (28). In fresh suspensions of rat proximal tubules, ANG IV inhibits energy-dependent transcellular Na+ transport by direct activation of AT4 receptors (28), and, in an immortalized human proximal tubular cell line, ANG IV stimulates expression of plasminogen activator inhibitor-1 (23). The physiological functions of ANG IV in proximal tubule and the intracellular signaling mechanisms associated with AT4 receptors remain incompletely understood. Accordingly, in the present studies we utilized a well-characterized proximal tubule-like cell line, LLC-PK1/Cl4, to determine signaling responses mediated by ANG IV. Abundant, specific binding of ANG IV was detected in these cells, with no effect of ANG IV on cellular levels of cAMP, cGMP, calcium, or on DNA synthesis. However, ANG IV causes a significant stimulation of tyrosine phosphorylation of two focal adhesion-associated proteins, p125 focal adhesion kinase (p125-FAK), and paxillin.


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Reagents and antibodies. 1:1 Bicarbonate-buffered mixture of DMEM and Ham's F-12 nutrient medium (DMEM/F-12) was purchased from GIBCO-BRL (Burlington, ON, Canada). ANG IV and ANG II were from Peninsula Laboratories (San Carios, CA). Losartan was generously provided by DuPont-Merck Pharmaceutical (Wilmington, DE), and PD-123319 was provided by Parke-Davis Pharmaceutical Research (Ann Arbor, MI). Fura 2-acetoxymethyl ester (fura 2-AM) and Pluronic-F127 were obtained from Molecular Probes, (Eugene, OR). Polyclonal and monoclonal (PY-20) anti-PY antibodies were purchased from Zymed Laboratories (San Francisco, CA). Monoclonal anti-paxillin and anti-FAK antibodies were from Transduction Laboratories (Lexington, KY). Polyclonal rabbit anti-FAK, anti-paxillin antibodies, and Protein A/G PLUS-Agarose beads were from Santa Cruz Biotechnology (Santa Cruz, CA). [125I]-labeled ANG IV (specific activity: 2,000 Ci/mmol), [125I]-labeled ANG II (specific activity: 2,200 Ci/mmol), the cGMP assay kit (containing [3H]cGMP), and enhanced chemiluminescence Hyperfilm were from Amersham Canada (Oakville, ON, Canada). The cAMP assay kit (containing [3H]cAMP) was from Intermedico, Markham, ON, Canada. All other chemicals were from Sigma (St. Louis, MO).

Cell culture. LLC-PK1/Cl4, an established proximal tubule epithelial cell line derived from pig kidney (1), was routinely cultured as previously described (11).

Ligand binding studies on cultured cells. LLC-PK1/Cl4 cells were seeded at a density of 2 × 104 cells/well in 24-well plastic plates. The cells were allowed to grow to subconfluence and made serum free 14-16 h before study. The cells were initially washed three times with PBS supplemented with 0.1% BSA and then exposed to 0.1 nM [125I]-ANG IV or [125I]-ANG II, in the presence or absence of unlabeled ligands at selected concentrations, in PBS-0.1% BSA containing EDTA (5 mM) and phenanthroline (1.25 mM) at 37°C for 1 h, followed by four washes with ice-cold PBS plus 0.1% BSA. After lysis in 0.05 M NaOH, cell-associated radioactivity was measured in a gamma counter (Beckman 5500B). Specific binding was determined by subtracting the nonspecific binding component measured in the presence of 10-6 M unlabeled ANG IV or ANG II from total [125I]-ANG IV or [125I]-ANG II binding, respectively. To estimate maximal binding capacity and the dissociation constant of AT4 receptors, saturation binding curves were generated by incubating the cells with increasing concentrations of [125I]-ANG IV (0.01-4 nM), in the presence or absence of 10-6 M unlabeled ANG IV. Protein concentration was measured with the Bio-Rad protein assay kit (Bio-Rad, Montreal, Canada) according to the manufacturer's instructions.

Determination of cAMP and cGMP concentrations. LLC-PK1/Cl4 cells were grown to confluence on 24-well dishes and then incubated for 16 h in serum-free DMEM/F-12 medium. For assays of cAMP or cGMP, cells were incubated at 37°C for 15 min in DMEM/F-12, supplemented with the phosphodiesterase inhibitor, IBMX (0.5 mM) and 0.5% BSA, in the presence or absence of ANG IV or other agonists. Medium was then aspirated and replaced with ice-cold 10% TCA (vol/vol). After 30 min, samples were extracted four times with 4 vol of water-saturated ether and brought to pH 7 with Tris. Aliquots were assayed for cAMP or cGMP, using radioligand competitive binding assay kits, as we have performed (6, 7).

Measurements of cytosolic calcium concentration ([Ca2+]i) in LLC-PK1/Cl4 cells. Subconfluent cells grown on round glass coverslips were serum starved for 24 h and loaded for 45 min at 37°C with 5 µM fura 2-AM in the presence of 0.005% Pluronic-F127 in serum-free DMEM. The coverslip was mounted in a thermally controlled chamber and continuously perfused with a buffer containing (in mM) 105 NaCl, 24 NaHCO3, 5 KCl, 2 Na2HPO4, 1 MgSO4, 1.5 CaCl2, 4 lactic acid, 5 glucose, 1 alanine, and 10 HEPES (pH 7.3) as well as 0.2% BSA. Fluorescence was measured from dual monochromators set at 340 and 380 nm, using a computer-linked analytical system (Photon Technology International, South Brunswick, NJ), coupled to a Nikon Diaphot TMD-inverted fluorescence microscope equipped with a CF ×100 objective (Nikon, Tokyo, Japan). Single cells or clusters of two to three cells were studied with a ×100 oil-immersion lens. The emissions at the excitation wavelengths of 340 and 380 nm were collected by a photomultiplier at 510 nm and used to calculate the fluorescence ratio (R340/380), which was used as an indicator of [Ca2+]i.

[3H]thymidine incorporation assay. Subconfluent cells in 24-well plates were made quiescent with serum-free medium. Agents were routinely added to the quiescent cells in triplicate or duplicate and incubated for 16 h, followed by the addition of 2 µCi/ml of [3H]thymidine to pulse the cells for an additional 2 h. Cells were then washed five times with ice-cold PBS plus 0.5% BSA and incubated with ice-cold 10% TCA, twice for 30 min each time. The TCA precipitates were washed with ice-cold 100% ethanol and then lysed in 0.25 N NaOH-0.1% SDS, and DNA-incorporated thymidine was counted by liquid scintillation spectrometry (11). Results were plotted as the number of counts per minute per well. Each experimental data point represents the mean value from triplicate or duplicate wells.

Immunoprecipitation and immunoblotting. Immunoprecipitation and immunoblotting procedures were performed as previously described (10). Briefly, subconfluent LLC-PK1/Cl4 cells were made quiescent and treated with the indicated agents, washed twice with ice-cold Ca2+/Mg2+-free PBS, and lysed on ice for 30 min in a lysis buffer containing 0.5% Nonidet P-40, 50 mM NaCl, 10 mM Tris · HCl (pH 7.4), 2 mM EDTA, 2 mM EGTA, 0.5% sodium deoxycholate, 0.1% SDS, 100 µM Na3VO4, 100 mM NaF, 30 mM sodium pyrophosphate, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, and 10 µg/ml leupeptin. Cell lysates were clarified at 10,000 g for 15 min at 4°C, and protein concentrations were measured. Target proteins were immunoprecipitated at 4°C for 2 h with appropriate antibodies. Immune complexes were captured with Protein A/G PLUS-Agarose beads, washed four times with wash buffer, and eluted by boiling in sample buffer, as previously described (12).

Immunoprecipitates were subjected to 7-15% SDS-PAGE, transferred onto polyvinylidene difluoride membranes, probed with the indicated primary antibody and the appropriate secondary antibody conjugated with biotin, and incubated with preformed avidin-biotin-horseradish peroxidase complex using a commercially available kit (ABC kit; Intermedico, Markham, ON), and the immune complexes were detected by a peroxidase-catalyzed enhanced chemiluminescence detection system.

Statistics. Data are presented as means ± SE for at least three separate experiments (each in triplicate or duplicate). Unpaired Student's t-test was used for statistical analysis. For multiple group comparisons, ANOVA and Bonferroni t-tests were used. A value of P < 0.05 was considered statistically significant.


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Binding studies. In LLC-PK1/Cl4 cells at 37°C, ANG IV dose dependently competed for [125I]-labeled ANG IV binding, and 10-6 M ANG IV almost completely abolished [125I]-labeled ANG IV binding (98.8% displacement) (Fig. 1A). In cells incubated with [125I]-labeled ANG IV for 16 h at 4°C, unlabeled ANG IV (10-6 M) still caused significant displacement of binding (88.5 ± 1.3% displacement; n = 4). At 4°C, saturation of specific binding was observed after ~4 h. Displacement binding experiments were performed further at 37°C to determine the ligand specificity of the receptors. ANG II (10-6-10-8 M) did not significantly displace [125I]-ANG IV binding, and neither the AT1 receptor antagonist, losartan (10-5-10-7 M), nor the AT2 receptor antagonist, PD-123319 (10-5-10-7 M) displaced [125I]-ANG IV binding (Fig. 1A). There was virtually no specific binding of [125I]-ANG II in these cells (n = 6, data not shown).


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Fig. 1.   Binding characteristics of [125I]-ANG IV in intact renal proximal tubular epithelial LLC-PK1/Cl4 cells. A: displacement of [125I]-ANG IV binding in LLC-PK1/Cl4 cells with increasing concentrations of unlabeled ANG IV, ANG II, losartan, and PD-123319. Data are expressed as percentages of maximal binding. Each point represents the mean ± SE of data from 4 separate experiments. B: saturation binding of [125I]-ANG IV in LLC-PK1/Cl4 cells. Inset: Scatchard plot analysis; these data describe binding sites of uniform high affinity for 125 I-ANG IV [dissocation constant (Kd) and maximal number of binding sites (Bmax) were 1.4 nM and 609.6 fmol/mg protein, respectively].

As shown in Fig. 1B, saturation binding studies indicated the presence of binding sites of uniform high affinity, with a dissociation constant of 1.4 nM and maximal amount of binding sites of 609.6 fmol/mg protein, derived from Scatchard analysis of the saturation binding data.

ANG IV does not increase [Ca2+]i in LLC-PK1/Cl4 cells. ANG IV has been shown to cause a transient rise in [Ca2+]i in cultured opossum kidney cells (17) and cultured rat mesangial cells (9), and recently, Handa et al. (27) reported that ANG IV induced a sustained elevation in [Ca2+]i in Mardin-Darby bovine kidney epithelial cells. In fura 2-AM-loaded LLC-PK1/Cl4 cells, ANG IV had no effect on [Ca2+]i, although exposure of the cells to arginine vasopressin (AVP, 10-6 M), used as a positive control, caused increases in [Ca2+]i (Fig. 2).


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Fig. 2.   Effect of ANG IV on cytosolic calcium concentration ([Ca2+]i) in LLC-PK1/Cl4 cells. Representative tracing showing absence of effect of ANG IV (10-6 M) on [Ca2+]i. Arginine vasopressin (AVP; 10-6 M), used as a positive control, caused a marked increase in [Ca2+]i in each of 5 separate experiments, whereas ANG IV had no effect on [Ca2+]i at concentrations between 10-6-10-8 M (n = 5).

Effects of ANG IV on cAMP and cGMP levels in LLC-PK1/Cl4 cells. ANG IV has been shown to stimulate cAMP production by human collecting duct cells in the presence of forskolin (15), and Patel et al. (34) have shown that ANG IV increased cGMP production in porcine pulmonary arterial endothelial cells. In LLC-PK1/Cl4 cells, ANG IV had no significant effect on either cAMP or cGMP levels, with positive controls (AVP for cAMP, atrial natriuretic peptide, and sodium nitroprusside for cGMP) demonstrating significant increases in levels of these second messengers (Fig. 3).


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Fig. 3.   Effects of ANG IV on cAMP and cGMP levels in LLC-PK1/Cl4 cells. A: absence of effect of 15-min incubation with ANG IV (10-7 M) on cAMP production. AVP (10-7 M) caused a significant increase in cAMP. Values are means ± SE. * P < 0.001 vs. control (C), n = 4. B: absence of effect of 15-min incubation with ANG IV (10-7 M) on cGMP production. Atrial natriuretic peptide (ANP; 10-6 M) or sodium nitroprusside (SNP; 10-4 M) caused significant increases in cGMP. Values are means ± SE. * P < 0.01 vs. control (C), ** P < 0.001 vs. C, n = 3.

Effect of ANG IV on [3H]thymidine incorporation in LLC-PK1/Cl4 cells. It is well established that ANG II-induced activation of AT1 receptors promotes proliferation in a number of cell types (43, 46). In contrast, recent studies suggest that AT2 receptors may inhibit cell growth, or cause apoptosis (19). To characterize the potential physiological roles of ANG IV in proximal tubule cells, we examined the effect of ANG IV on DNA synthesis in LLC-PK1/Cl4 cells. ANG IV (10-7 M) did not stimulate [3H]thymidine incorporation in quiescent cells (control: 11,104 ± 2,036 vs. ANG IV: 11,023 ± 1,950 dpm/well; n = 5, P = NS).

ANG IV induces protein tyrosine phosphorylation. Protein tyrosine phosphorylation represents a common mechanism of growth control in many cells. To examine if ANG IV could activate protein tyrosine kinase cascades after binding to its AT4 receptor, confluent LLC-PK1/Cl4 cells were rendered quiescent by incubation in serum-free medium for 72 h, and then treated with ANG IV (10-7 M) for different time periods. After solubilization, cell lysates were subjected to immunoprecipitation with polyclonal anti-phosphotyrosine (PY) and immunoblotting with monoclonal anti-phosphotyrosine (PY-20). Administration of ANG IV stimulated marked increases in tyrosine phosphorylation of several proteins, with two predominant bands, one at ~125 kDa, and the other at ~68 kDa. This effect was observed within 2 min, and peaked at 30 min after exposure to 10-7 M ANG IV (Fig. 4).


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Fig. 4.   ANG IV increases tyrosine phosphorylation in a time-dependent manner. Quiescent LLC-PK1/Cl4 cells were treated with vehicle (H2O) alone or ANG IV (10-7 M) for different periods of time as indicated, and tyrosine-phosphorylated proteins were immunoprecipitated with polyclonal anti-phosphotyrosine (anti-PY) antibodies and immunoblotted with monoclonal anti-phosphotyrosine (PY-20) antibodies as described in MATERIALS AND METHODS. IP, immunoprecipitation; IB, immunoblotting.

To determine the lowest effective concentration of ANG IV to stimulate tyrosine phosphorylation, quiescent LLC-PK1/Cl4 cells were treated with different concentrations of ANG IV (10-6 to 10-11 M) for 15 min. ANG IV-stimulated protein tyrosine phosphorylation was dose dependent, first detectable at 10-9 M, and reached maximal induction at 10-7 M (Fig. 5), results consistent with effects due to functional coupling of ANG IV receptors.


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Fig. 5.   Increases in protein tyrosine phosphorylation in response to different concentrations of ANG IV. Quiescent LLC-PK1/Cl4 cells were treated with or without different concentrations of ANG IV (10-6-10-11 M) as indicated, and tyrosine-phosphorylated proteins were immunoprecipitated with polyclonal anti-PY antibodies and immunoprobed with PY-20.

Immunoprecipitation with an antibody to p125-FAK, followed by immunoblotting with anti-PY antibody (or immunoprecipitation with anti-PY antibodies followed by immunoblotting with anti-FAK) demonstrated that the tyrosine-phosphorylated protein of ~125 kDa was indeed p125-FAK (Fig. 6). The tyrosine phosphorylation of p125-FAK by ANG IV occurred with the same time course as in tyrosine phosphorylation immunoblotting experiments (Fig. 4), with increased tyrosine phosphorylation of p125-FAK within 2 min after ANG IV addition, peaking at 30 min, and present for at least 60 min. Administration of ANG II (10-7 M) had no effect on tyrosine phosphorylation of p125-FAK in LLC-PK1/Cl4 cells, and pretreatment of cells with either losartan (10-6 M) or PD-123319 (10-6 M) did not alter ANG IV-induced tyrosine phosphorylation of p125-FAK (Fig. 7).


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Fig. 6.   ANG IV stimulates tyrosine phosphorylation of p125-focal adhesion kinase (FAK) in a time-dependent manner. After treatment with or without ANG IV (10-7 M), cell lysates were immunoprecipitated with an anti-FAK antibody and immunoprobed with a monoclonal anti-PY antibody (top), and then the membrane was stripped and reprobed with anti-FAK antibodies to ensure that equivalent amounts of total FAK were immunoprecipitated in each sample (bottom).



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Fig. 7.   Effect of ANG II alone on tyrosine phosphorylation of p125-FAK, and effect of losartan or PD-123319 on ANG IV-induced tyrosine phosphorylation of p125-FAK. Quiescent LLC-PK1/Cl4 cells were treated with ANG II (10-7 M) alone, or ANG IV (10-7 M) with or without losartan (10-6 M) or PD-123319 (10-6 M) pretreatment. After IP with anti-FAK antibodies, and IB with a monoclonal anti-PY antibody (top), the blot was stripped and reprobed with anti-FAK antibodies to ensure that equivalent amounts of total FAK sample were immunoprecipitated in each sample (bottom panel).

Immunoprecipitation with anti-PY antibody and immunoblotting with a monoclonal anti-paxillin antibody identified the tyrosine-phosphorylated protein of ~68 kDa as p68-paxillin. Increases in p68-paxillin tyrosine phosphorylation were noted within the first 2 min of ANG IV addition, peaked at 30 min, and decreased at 60 min (Fig. 8). As with p125-FAK, ANG II (10-7 M) did not stimulate p68-paxillin tyrosine phosphorylation, and ANG IV-stimulated tyrosine phosphorylation of p68-paxillin was not affected by pretreatment of cells with either losartan (10-6 M) or PD-123319 (10-6 M) (Fig. 9), suggesting that the effects of ANG IV are mediated via activation of AT4 receptors.


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Fig. 8.   ANG IV stimulates tyrosine phosphorylation of paxillin in a time-dependent manner. Quiescent LLC-PK1/Cl4 cells were exposed to ANG IV (10-7 M) or vehicle (H2O) for the indicated times. Cell lysates were subjected to IP with polyclonal anti-PY antibodies and immunoprobing with a monoclonal anti-paxillin antibody. Tyrosine-phosphorylated paxillin (68 kDa) is indicated (arrow).



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Fig. 9.   Effect of ANG II alone on tyrosine phosphorylation of p68-paxillin, and effect of losartan or PD-123319 on ANG IV-stimulated tyrosine phosphorylation of paxillin. Quiescent LLC-PK1/Cl4 cells were treated with ANG II (10-7 M) alone, or ANG IV (10-7 M) with or without pretreatment of the cells with losartan (10-6 M) or PD-123319 (10-6 M). Cell lysates were immunoprecipitated with polyclonal anti-PY antibodies and immunoprobed with a monoclonal anti-paxillin antibody as described in MATERIALS AND METHODS. The 68 kDa tyrosine-phosphorylated paxillin is indicated (arrow).


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
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Specific binding sites for ANG IV have been identified in several mammalian tissues, including brain, spinal cord, colon, heart, lung, bladder, adrenal gland, and kidney, often by [125I]-ANG IV binding assays on isolated cell membranes (29, 31, 34). In the present study, we performed [125I]-ANG IV binding in intact LLC-PK1/Cl4 cells, a porcine renal proximal tubule cell line expressing no detectable ANG II AT1 and AT2 receptors (3, 6), and identified the presence of AT4 receptors of high affinity, with high specificity for ANG IV. Our data do not exclude the possibility of receptor subtypes for ANG IV in these cells of uniform affinity. These receptors have a maximal amount of binding sites of 609.6 fmol/mg of cellular proteins, reflecting a high concentration of AT4 receptors in these cells. Therefore, this is an ideal cell model to investigate the potential physiological functions and mechanisms of action of ANG IV.

To study AT4 receptor-mediated intracellular signaling mechanisms in these cells, we examined a number of classical second messengers. Our results indicate that ANG IV had no effect on [Ca2+]i or on intracellular levels of cAMP or cGMP in these cells. In certain tissues, ligand binding to plasma membrane AT4 receptors has been shown to increase [Ca2+]i (16, 17, 27). Enhanced cAMP production (15), and elevated nitric oxide synthase activity leading to increased cGMP production, have also been reported to be induced by AT4 receptor activation (34). This suggests that after ANG IV binding, activation of AT4 receptors initiates intracellular signaling events in a cell type- or species-specific fashion. This notion is supported by the observation that ANG IV did not exert any effect on [Ca2+]i in embryonic chick cardiac myocytes (2) or neuroblastoma cells (36), and ANG IV did not alter either [Ca2+]i or cGMP production in human collecting duct cells (15). Similarly, ANG IV had no effect on [Ca2+]i or cAMP production in bovine aortic endothelial cells (5). Although our data revealed no significant effect of ANG IV on DNA synthesis in LLC-PK1/Cl4 cells, a cell growth-promoting effect of ANG IV has been observed in other cell types (25, 35, 47), although the mitogenic signaling mechanisms of ANG IV remain undefined.

Protein tyrosine phosphorylation by activation of tyrosine kinases is one of the most common and important regulatory mechanisms in signal transmission. Accordingly, we examined the possibility that AT4 receptors might be linked to tyrosine kinase phosphorylation pathways. The present studies are the first demonstration that binding of ANG IV to its AT4 receptor stimulates marked increases in tyrosine phosphorylation of the focal adhesion-associated proteins, p125-FAK and paxillin, in a time- and concentration-dependent manner. In the radioligand binding studies, 10-7 M ANG IV almost completely abolished [125I]-labeled ANG IV binding, and ANG IV-stimulated tyrosine phosphorylation was detectable at 10-9 M, and reached a maximum at 10-7 M, with no additional stimulation at 10-6 M. This effect of ANG IV was not altered by the AT1 receptor antagonist, losartan or the AT2 receptor antagonist, PD-123319. In addition, the precursor of ANG IV, ANG II, had no significant effect on tyrosine phosphorylation of either p125-FAK or paxillin, and, indeed, there was no specific binding of [125I]-ANG II in these cells. Together, these results indicate that ANG IV induces tyrosine phosphorylation of these two focal adhesion-associated proteins via functional coupling to specific AT4 receptors.

Our study did not focus on the upstream or downstream signaling pathways of p125-FAK or paxillin in the ANG IV-induced tyrosine kinase cascade in LLC-PK1/Cl4 cells. In this regard, p125-FAK and paxillin have been shown to colocalize with integrins, transmembrane receptors that engage extracellular matrix ligands such as fibronectin, to the focal adhesion complex, where p125-FAK plays a central role in the regulation of cell signaling, proliferation, apoptosis, migration, spreading, and oncogenic transformation (8, 41). p125-FAK associates with a number of signaling and cytoskeletal molecules, including Src kinases, PI 3-kinase, Shc, Grb2, p130Cas (crk-associated substrate), talin, and paxillin (41). On integrin-dependent cell adhesion, p125-FAK becomes autophosphorylated on Tyr-397, creating a high affinity Src SH2-binding site, and becomes enzymatically activated (18, 38). On binding to Tyr-397 of p125-FAK, Src can phosphorylate p125-FAK on a number of additional tyrosine residues, thereby resulting in enhanced catalytic activity of p125-FAK and inducing the formation of docking sites on p125-FAK for additional SH2-containing binding partners, such as Grb2 (40). Formation of the p125-FAK-Src complex can direct tyrosine phosphorylation of additional focal adhesion-associated substrates such as paxillin and p130Cas, which results in the recruitment of additional SH2 domain-containing signaling molecules such as Crk (39, 41). Thus Src acts upstream of p125-FAK and plays an essential role in p125-FAK tyrosine phosphorylation of paxillin and p130Cas in these complexes. In addition, although its predominant role is in integrin signal transduction, p125-FAK is also tyrosine phosphorylated in response to a variety of other extracellular stimuli that act either on specific membrane receptors or on intracellular signaling molecules, such as protein kinase C or Rho (8, 37, 41), and initiation of the Ras/mitogen-activated protein kinase pathway has been suggested to be a downstream signaling event of p125-FAK activation (40, 41). Therefore, it is reasonable to speculate that binding of ANG IV to AT4 receptors in LLC-PK1/Cl4 cells might activate Src kinases and induce p125-FAK tyrosine phosphorylation, which then leads to tyrosine phosphorylation of paxillin, as well as extracellular signal-related kinase. Phosphorylation of p125-FAK and paxillin by ANG IV also suggests the possibility that ANG IV might influence a range of physiological parameters in proximal tubule cells, including cell adhesion, cell spreading, and cell motility.

Further studies characterizing the structure of the AT4 receptor should provide insight into these signaling pathways and the biological effects of ANG IV. In this regard, it has been suggested that unlike the AT1 or AT2 receptor, the AT4 receptor is not a G protein-coupled receptor, because guanosine 5'-O-(3-thiotriphosphate)gamma S and polyvinyl sulfate do not affect [125I]-ANG IV binding (4, 27). Cross-linking and photoaffinity-labeling studies suggest that the AT4 receptor is a ~165-186 kDa integral membrane glycoprotein with a very large extracellular domain, a single transmembrane domain, and a short intracellular tail (4, 5, 51). These properties, together with our demonstration of activation of tyrosine phosphorylation pathways, suggest that the AT4 receptor may be a member of the family of growth factor or cytokine receptors, which includes receptors for epidermal growth factor (45), interleukin-11 (33), and granulocyte-macrophage colony-stimulating factor (22).

The generation of ANG IV has been thought to occur via successive cleavage of ANG II by aminopeptidases A and N. Recently, a novel human zinc metalloprotease has been cloned, representing a homolog of ACE (44). This novel protein is highly expressed in kidney, heart, and testis and acts as a carboxypeptidase to cleave the COOH-terminal residues from ANG I and ANG II, forming ANG-(1-9) and ANG-(1-7), respectively. The role of this enzyme in regulating production of ANG IV in the proximal tubule remains to be clarified. Furthermore, the possibility of functional coupling of other locally generated peptide fragments to ANG IV receptors requires further study. In this regard, ANG-(1-7) has been shown to bind to ANG IV receptors in proximal tubule cells (26), although effects on protein tyrosine phosphorylation have not been reported.

In summary, we have demonstrated that LLC-PK1/Cl4 cells express functional AT4 receptors of uniform high affinity, and ligand-dependent activation of these receptors induces tyrosine phosphorylation of focal adhesion-associated proteins. The present studies suggest that in addition to activation of AT1 or AT2 receptors, ANG II may modulate function of the focal adhesion complex by metabolism to ANG IV and activation of AT4 receptors.


    ACKNOWLEDGEMENTS

This work was supported by a grant from the Kidney Foundation of Canada (to K. D. Burns).


    FOOTNOTES

Address for reprint requests and other correspondence: K. D. Burns, Div. of Nephrology, Univ. of Ottawa and The Ottawa Hospital, 501 Smyth Rd., Rm. LM-18, Ottawa, Ontario, Canada K1H 8L6 (E-mail: kburns{at}ottawahospital.on.ca).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 28 September 2000; accepted in final form 26 January 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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