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(Hypertension. 1997;30:351.)
© 1997 American Heart Association, Inc.

Articles

Angiotensin II–Induced Phosphorylation of the AT₁ Receptor From Rat Brain Neurons

Hong Yang; Di Lu; Mohan K. Raizada

From the Department of Physiology, College of Medicine, University of Florida, Gainesville.

Correspondence to Mohan K. Raizada, PhD, Professor and Associate Dean for Graduate Education, Department of Physiology, College of Medicine, University of Florida, PO Box 100274, Gainesville, FL 32610. E-mail mraizada{at}phys.med.ufl.edu

	Abstract

Top Abstract Introduction Methods Results Discussion References

 
Abstract The neuronal angiotensin II (Ang II) type 1 (AT₁) receptor is coupled to the Ras-Raf-1–mitogen-activated protein (MAP) kinase signal-transduction pathway (Yang H, Lu D, Yu K, Raizada MK. Regulation of neuromodulatory actions of angiotensin II in the brain neurons by the Ras-dependent mitogen-activated protein kinase pathway. J Neurosci. 1996;16:4047-4058). In this study we compared the effects of angiotensin II (Ang II) on AT₁ receptor phosphorylation and the ability of the phosphorylated receptor to bind Ang II in neuronal cultures of Wistar-Kyoto rat (WKY) and spontaneously hypertensive rat (SHR) brains to further our understanding of the Ang II signaling mechanism. Ang II caused a time-dependent phosphorylation of AT₁ receptors in both WKY and SHR brain neurons. The level of phosphorylation was higher in the SHR brain neurons; this finding was consistent with increased AT₁ receptors in these cells. MAP kinase was involved in this phosphorylation, a conclusion supported by the following evidence: (1) exogenous MAP kinase phosphorylated the AT₁ receptor; (2) PD98059, a MAP kinase kinase inhibitor, attenuated Ang II–stimulated AT₁ receptor phosphorylation; and (3) MAP kinase and AT₁ receptors were coimmunoprecipitated in Ang II–stimulated neurons. Finally, MAP kinase phosphorylation was associated with the loss of ¹²⁵I-[Sar¹-Ile⁸]-Ang II binding ability of the AT₁ receptor in both strains of neurons. These observations show that Ang II stimulates phosphorylation of the neuronal AT₁ receptor by a mechanism involving MAP kinase and that the phosphorylated neuronal AT₁ receptor does not exhibit Ang II binding activity in the brains of either WKY or SHR.

Key Words: receptors, angiotensin II • MAP kinase • phosphorylation • angiotensin II • rats, inbred SHR • rats, inbred WKY

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
Angiotensin II stimulates tyrosine hydroxylase, dopamine ß-hydroxylase, and NET by activating the AT₁ receptor subtype in neuronal cultures.^{1 2} The neuromodulatory actions of the AT₁ receptor, a member of the GPCR superfamily, are associated with the regulation of turnover, synthesis, and release of catecholamines in brain neurons.^{3 4} The physiological relevance of these actions of Ang II in brain neurons is further evidenced by observations that neurons from SHR brain express higher levels of AT₁ receptors and a parallel increase in the Ang II–induced NET system.^{2 4 5 6 7} In recent studies, we have established that Ang II stimulation of neuromodulation involves activation of the Ras-Raf-1–MAP kinase signal-transduction pathway.⁸ Activation of MAP kinase initiates a cascade of downstream signaling events involving SRE and AP₁ binding activities that ultimately leads to increased gene expression for tyrosine hydroxylase, dopamine ß-hydroxylase, and NET.⁹

Despite our understanding of the signal-transduction pathways involved in the cellular and physiological actions of the AT₁ receptor in neurons, little is known about the mechanism by which these signals are terminated. Studies with other GPCRs have indicated that an agonist-induced phosphorylation of the GPCR plays a key role in the desensitization of the receptor and termination of physiological actions.^{10 11} Many specific GRKs have been characterized in recent years that specifically phosphorylate agonist-bound GPCRs.^{10 11} In view of the potential role of phosphorylation in the regulation of the functions of GPCRs, the present study was conducted with the following two objectives in mind: (1) to determine whether Ang II stimulates phosphorylation of the AT₁ receptor and if so, whether MAP kinase is involved in this phosphorylation; and (2) to compare the rates and mechanisms of Ang II–induced phosphorylation of AT₁ receptors in neurons of WKY and SHR brains. In view of our previous observation that both AT₁ receptors and AT₁ receptor-mediated neuromodulation are increased in SHR brain neurons,^{4 5} we hypothesized that the rate of phosphorylation of the AT₁ receptor would either not change or be decreased in these neurons compared with their normotensive WKY counterparts. Our observations show that Ang II stimulates AT₁ receptor phosphorylation in the neurons of both strains of rats, an effect mediated by the activation of MAP kinase. The phosphorylation rate of the AT₁ receptor in both WKY and SHR brain neurons was comparable.

	Methods

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One-day-old WKY and SHR were obtained from our breeding colony, which originated from Harlan Sprague Dawley (Indianapolis, Ind). DMEM, PDHS, and trypsin (150 IU/mg) were from Central Biomedia. [-³²P]ATP (3000 Ci/mmol), [³²P]orthophosphate (1 mCi=37 MBq), and chemiluminescence assay reagents were from Du Pont/NEN. Nitrocellulose membranes were from Micron Separations, Inc. Ang II and MBP were purchased from Sigma Chemical Co. Losartan potassium (Dup 753) was a gift from Du Pont/Merck (Wilmington, Del). PD98059, H89, bis-indolylmaleimide, and lavendustin A were purchased from Calbiochem. PD123319 was from RBI. Polyclonal anti-rabbit AT₁ receptor antibody (306,Sc-579) was purchased from Santa Cruz Biotechnology (catalog No. 579). This antipeptide antibody was raised against an AT₁ receptor peptide corresponding to amino acids^306-359. The antibody was specific for the AT₁ receptor, did not cross-react with the AT₂ receptor, and was mouse, rat, and human reactive. Anti-MAP kinase (C-14) polyclonal antibody that specifically recognizes ERK-2 (and to a much lesser extent, ERK-1) and protein A/G PLUS–agarose were purchased from Santa Cruz Biotechnology. An anti-rat MAP kinase polyclonal antibody (ERK-1 through ERK-3), which recognized both the p42 and p44 isoforms of MAP kinase, and activated mouse GST-p42 MAP kinase were from Upstate Biotechnology, Inc. All other reagents were purchased from Fisher Scientific and were of the highest quality available.

Hypothalamus-Brainstem Neuronal Cells in Primary Culture
Hypothalamus-brainstem areas of 1-day-old WKY and SHR brains were dissected and brain cells were dissociated by trypsin. The hypothalamic block contained the paraventricular nucleus and the supraoptic, anterior, lateral, posterior, dorsomedial, and ventromedial nuclei. The brainstem block contained the medulla oblongata and pons. Dissociated brain cells were plated in poly-L-lysine–precoated tissue-culture dishes (2x10⁷ cells per 100-mm-diameter dish) in DMEM containing 10% PDHS. Neuronal cultures were established essentially as previously described.^{5 12} The cultures were allowed to grow for 15 days before use in experiments. Immunohistochemical and biochemical analyses have repeatedly indicated that these cultures contain 90% neuronal cells and 15% astroglial cells and that cultures are comparable from the two strains of rats.^{5 12}

Immunoprecipitation
Neuronal cultures, established in 100-mm culture dishes, were treated with Ang II. The cell lysates were prepared by adding 1 mL lysis buffer (25 mmol/L Tris-HCl, pH 7.4; 25 mmol/L NaCl; 1% Triton X-100; 1% deoxycholic acid; 1 mmol/L sodium orthovanadate; 10 mmol/L sodium fluoride; 10 mmol/L sodium pyrophosphate; 0.5 mmol/L EGTA; 1 mmol/L PMSF; 10 µg/mL aprotinin; and 0.8 µg/mL leupeptin) and scraping the cells off the culture dish. The lysates were centrifuged at 6000g for 10 minutes, and the protein content of the supernatant was assessed by the Bradford protein assay (Bio-Rad Laboratories). Supernatants containing 400 µg protein were subjected to an immunoprecipitation protocol as follows. Samples were incubated with 1 µg rabbit anti-AT₁ receptor antibody overnight at 4°C, and AT₁ receptor immunoreactivities were collected on protein A/G PLUS–agarose.^{8 13} The resulting immunoprecipitates were washed three times with lysis buffer and used in further experiments.

Immunoblotting
Each immunoprecipitate was suspended in 20 µL Laemmli’s sample buffer in a boiling water bath for 3 minutes, then centrifuged. The resulting supernatant (10 µL) was electrophoresed in 10% SDS–polyacrylamide gel and proteins were transferred onto a nitrocellulose membrane. The membrane was blocked by 5% nonfat dry milk in TBST (20 mmol/L Tris-HCl, pH 8.0; 150 mmol/L NaCl; and 0.05% Tween 20) for 1 hour followed by incubation for 1 hour at room temperature with either rabbit anti-MAP kinase antibody or anti-AT₁ receptor antibody. Protein-bound antibody was detected by incubation of the membrane with horseradish peroxidase–labeled second antibody and enhanced by chemiluminescence assay reagents. The bands recognized by the primary antibody were visualized by exposing the membrane to x-ray film.⁸

Phosphorylation of AT₁ Receptor by Exogenous MAP Kinase
Phosphorylation of the AT₁ receptor, after its immunoprecipitation from the neuronal cell lysate by anti-rabbit AT₁ receptor antibody, was carried out with the use of exogenous MAP kinase in a protocol based on that described by Paxton et al.¹⁴ Briefly, neuronal cell lysates were prepared as described above. Lysate containing equal amounts of protein (400 µg) was incubated with 1 µg rabbit anti-AT₁ receptor antibody overnight at 4°C, and AT₁ receptor immunoprecipitate was collected on protein A/G PLUS–agarose. This preparation was rinsed three times with lysis buffer and once with kinase assay buffer (50 mmol/L HEPES, pH 7.5; 0.1 mmol/L EDTA; and 0.015% Triton X-100). Immunoprecipitate was suspended in 10 µL kinase assay buffer. For measurement of phosphorylation, 10 µL of AT₁ receptor immunoprecipitate (30 fmol/mg ¹²⁵I-[Sar¹-Ile⁸]-Ang II binding activity) was incubated with 0.3 IU MAP kinase, 0.1 mg/mL BSA, and 0.2% ß-mercaptoethanol in a final volume of 20 µL. The reaction was started by the addition of 10 µL ATP mixture (0.3 mmol/L ATP, 30 mmol/L MgCl, and 200 µCi [-³²P]ATP per milliliter in kinase assay buffer) and run for 0 to 15 minutes at 30°C. After the reaction was stopped by adding phosphoric acid, samples were blotted onto Whatman GF/B filter paper followed by washing the paper four times with ice-cold 0.5% phosphoric acid and finally once with acetone essentially as described elsewhere.¹⁴ The paper was allowed to dry and radioactivity quantitated in a Beckman liquid scintillation counter. Reaction mixtures that contained 3 µg MBP instead of AT₁ receptor immunoprecipitate were used as the standard for the MAP kinase phosphorylation assay. In some experiments, the kinase reaction was stopped by the addition of 5x Laemmli’s sample buffer instead of phosphoric acid; samples were heated and centrifuged, and the supernatant was electrophoresed in 10% SDS–polyacrylamide gel. Proteins were then transferred to polyvinylidene difluoride membrane and subjected to autoradiography. After autoradiography, the same membrane was used for immunoblotting with AT₁ receptor antibody. Autoradiograms were scanned using a UVP Imagestore 5000 system, and receptor phosphorylation data were presented as the observed density of the ³²P-labeled band that was normalized for AT₁ receptor protein by densitometry using the SW 5000 gel analysis program.

Labeling of Neuronal Cells With [³²P]Orthophosphate and Analysis of Phosphorylated AT₁ Receptor
Neuronal cultures were established for 15 days in 100-mm culture dishes. Growth medium was removed and cultures were incubated with phosphate-free DMEM containing dialyzed PDHS for 4 hours at 37°C, followed by prelabeling the cells with 1 mCi/mL [³²P]orthophosphate for 4 hours at 37°C. Ang II was added and incubation continued for various time periods. Cultures were immediately rinsed with ice-cold PBS three to four times, and lysates were prepared in lysis buffer as described above. Cell lysates were centrifuged at 6000g for 10 minutes and the supernatants used to immunoprecipitate AT₁ receptors essentially as described above. Agarose beads containing AT₁ receptor were collected at 3000g for 10 minutes, washed three times in lysis buffer, resuspended in 20 µL Laemmli’s sample buffer, and heated to 100°C for 3 minutes. The supernatant was electrophoresed in 10% SDS–polyacrylamide gel, and proteins were transferred to polyvinylidene difluoride membrane. The membrane was dried and subjected to autoradiography. Densities of the ³²P-labeled bands corresponding to AT₁ receptors were scanned with the use of the UVP Imagestore 5000 system and quantitated by the SW 5000 gel analysis program. Data were normalized to the densities of total AT₁ receptor in each lane as determined by immunoblotting.

Binding of ¹²⁵I-[Sar¹-Ile⁸]-Ang II to AT₁ Receptor Immunoprecipitates
Cell-free lysates (400 µg protein) were subjected to immunoprecipitation by rabbit anti-AT₁ receptor antibody as described above. Immunoprecipitates containing AT₁ receptor were collected on protein A/G PLUS–agarose and washed three times with lysis buffer and once with kinase buffer. Immunoprecipitates were subjected to phosphorylation by exogenous MAP kinase essentially as described above. Immune complexes were rinsed with the binding buffer (PBS, pH 7.2, containing 1.0% BSA) and used for quantitation of ¹²⁵I-[Sar¹-Ile⁸]-Ang II binding as described previously.¹⁵ In brief, immune complexes suspended in 0.5 mL binding buffer were incubated with 1 nmol/L ¹²⁵I-[Sar¹-Ile⁸]-Ang II in the presence of 10 µmol/L PD123319 for 1 hour at room temperature to determine total binding. In addition, increasing concentrations of losartan (0.01 nmol/L to 100 nmol/L) were used for competition-inhibition experiments. All reactions were run in triplicate. The binding reaction was terminated by filtration and collection of ¹²⁵I-[Sar¹-Ile⁸]-Ang II bound to receptors on Whatman GF/B filters presoaked with 0.3% polyethyleneimine. Filters were washed three times with ice-cold PBS, pH 7.2, to remove unbound radioligand, and bound radioactivity was counted by a Beckman DP5500 gamma counter. Binding was expressed as femtomoles ¹²⁵I-[Sar¹-Ile⁸]-Ang II bound per milligram of the cellular protein used to immunoprecipitate the receptor. Specific binding was calculated by subtracting the ¹²⁵I-[Sar¹-Ile⁸]-Ang II bound to complex in the presence of losartan from that bound in its absence. Scatchard analysis of the competition-inhibition experiments was conducted to calculate K_d and B_max values using the EBDA-ligand program (Elsevier-Biosoft).²⁶

Data Analysis
Each experiment was conducted in triplicate culture dishes. Cells in these dishes were derived from multiple brains of 1-day-old rats. Each experiment was repeated three times unless indicated otherwise. Images from autoradiograms were captured in the UVP Imagestore 5000 system, and radioactive bands were quantitated essentially as described elsewhere.⁸ Data from at least three autoradiograms were quantitated and corrected for equal loading with the use of AT₁ receptor antibody, ¹²⁵I-[Sar¹-Ile⁸]-Ang II binding activity to the AT₁ receptor, or another standard protein. They are presented as mean±SE. Statistical analysis was performed using ANOVA and Dunnett’s tests.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Previous studies have established that neuronal cultures of SHR brain express two to four times higher levels of AT₁ receptors than neuronal cultures of WKY brain.^{4 5} This increase is associated with an increased ability of Ang II to stimulate NET.² Our initial objective in this study was to compare the effect of Ang II on AT₁ receptor phosphorylation in these two strains of rat brain neurons. Prelabeling of neuronal cultures with [³²P]orthophosphate followed by stimulation with Ang II and immunoprecipitation analysis of ³²P-labeled AT₁ receptor showed a radiolabeled band of 49 kD in both WKY and SHR brain neurons (Fig 1A). Ang II caused a time-dependent increase in the incorporation of ³²P into this band, and a sixfold increase was observed in WKY brain neurons in 15 minutes. This increase was followed by 50% decrease in ³²P incorporation by 30 minutes. Basal levels of phosphorylation of ³²P into AT₁ receptors were 65% higher in SHR brain neurons than in WKY brain neurons in spite of a comparable time course of Ang II stimulation (Fig 1B). As a result, the degree of stimulation (approximately sixfold) of Ang II– induced AT₁ receptor phosphorylation was comparable in both strains. This conclusion was confirmed when AT₁ receptor phosphorylation data were normalized with the levels of AT₁ receptor immunoreactivity in both strains of neurons (Fig 1C). The 49-kD band is AT₁ receptor protein, on the basis of the ability of the AT₁ receptor-specific antibody to recognize this protein¹⁶ and the observation that the immunoprecipitated protein specifically binds ¹²⁵I-[Sar¹-Ile⁸]-Ang II (see discussion to follow). Fig 2 shows that Ang II–induced phosphorylation of neuronal AT₁ receptor was completely blocked by 10 µmol/L losartan but not by PD123319, an AT₂ receptor subtype antagonist. This observation suggested that the occupancy by Ang II is important for stimulation of the AT₁ receptor phosphorylation.

View larger version (19K): [in this window] [in a new window]   Figure 1. Ang II stimulation of AT1 receptor phosphorylation in WKY and SHR brain neuronal cultures. A, Neuronal cultures, established in 100-mm-diameter culture dishes, were prelabeled with [32P]orthophosphate for 4 hours and incubated with 100 nmol/L Ang II for the indicated time periods. Cellular protein (400 µg) containing AT1 receptors (equivalent to 30 fmol/mg for WKY neurons and 50 fmol/mg for SHR neurons of 125I-[Sar1-Ile8]-Ang II specific binding to AT1 receptors26 ) was immunoprecipitated and then subjected to SDS-PAGE and autoradiography as described in "Methods." A representative autoradiogram representing 32P-labeled AT1 receptor is shown. The membrane was also probed with AT1 receptor antibody to determine equal loading in each lane, as described in "Methods." B, Quantitation of the radioactive band representing 32P-labeled AT1 receptor from three separate experiments. Densitometric absorbance values from three separate experiments were averaged (±SE) after they were normalized for equal loading. C, Data from A normalized for equal amounts of AT1 receptor immunoreactivity for both WKY and SHR brain neurons.

View larger version (29K): [in this window] [in a new window]   Figure 2. Effect of Ang II receptor antagonists on AT1 receptor phosphorylation in WKY neuronal cultures. Neuronal cultures were prelabeled with [32P]orthophosphate for 4 hours and incubated without (1, 3, and 5) or with (2, 4, and 6) 100 nmol/L Ang II for 10 minutes in the absence (1 and 2) or presence (3 and 4) of 10 µmol/L losartan or 10 µmol/L PD123319 (5 and 6). Immunoprecipitation of 32P-labeled AT1 receptor was carried out essentially as described in the legend to Fig 1. AT1 receptor protein was used to normalize for equal loading. Data were analyzed essentially as described for Fig 1. Top, Representative autoradiogram. Bottom, Quantitation of 32P-labeled AT1 receptor after normalization with AT1 receptor immunoreactivity. Data are presented as fold increase, using control as the baseline. *Significantly different from 1 (P<.05). **Significantly different from 2 (P<.05).

Next we studied the involvement of MAP kinase in Ang II–stimulated phosphorylation of the AT₁ receptor. The rationale for this experiment was based on our previous studies, which showed that Ang II stimulates MAP kinase in neurons.⁸ In addition, MAP kinase has been implicated in the phosphorylation of estrogen and epidermal growth factor receptors.^{17 18} AT₁ receptors were immunoprecipitated from WKY and SHR brain neurons, and these immunoprecipitates were subjected to an in vitro phosphorylation assay with exogenous MAP kinase. Fig 3A shows that exogenous MAP kinase catalyzed the phosphorylation of immunoprecipitated AT₁ receptors in a time-dependent manner and that the phosphorylation in both strains of neurons was comparable. MBP was used as an internal standard for MAP kinase substrate. SDS-PAGE analysis of in vitro phosphorylated immunoprecipitated AT₁ receptors was carried out to confirm the identity of phosphorylated protein. Fig 3B shows that the 49-kD ³²P-labeled band was recognized by AT₁ receptor antibody on the immunoblot. In addition, incorporation of ³²P into this band increased over time in both WKY and SHR brain. This finding was consistent with the in vitro phosphorylation data (Fig 3A). These observations indicate that exogenous MAP kinase could phosphorylate the AT₁ receptor. Neuronal cultures were treated with PD98059, a relatively selective inhibitor of MAP kinase kinase, in an attempt to provide in vivo support for the above conclusion. Cultures were prelabeled with [³²P]orthophosphate for 4 hours followed by coincubation without or with Ang II in the presence of 10 µmol/L PD98059. This concentration of PD98059 has previously been shown to inhibit MAP kinase kinase activity.¹⁹ In addition, a 90% inhibition of neuronal MAP kinase activity using this treatment was observed. Fig 4 shows that PD98059 significantly inhibited Ang II–stimulated phosphorylation of AT₁ receptors in both WKY and SHR brain neurons. Other protein kinase inhibitors were used to further establish the specificity of MAP kinase in Ang II–induced phosphorylation of the AT₁ receptor. Fig 5 shows that basal AT₁ receptor phosphorylation was not affected to any significant degree by inhibitors of protein kinase A (H89) and protein tyrosine kinase (lavendustin). However, Ang II stimulation of AT₁ receptor phosphorylation was partially blocked by the PKC inhibitor bis-indolylmaleimide. This observation was consistent with the report that PKC phosphorylation sites are present in the AT₁ receptor and that Ang II stimulates neuronal PKC.^{20 21} Inhibition of protein tyrosine kinase or protein kinase A had little effect on phosphorylation.

View larger version (17K): [in this window] [in a new window]   Figure 3. A, In vitro phosphorylation of AT1 receptors by MAP kinase. AT1 receptors were isolated by immunoprecipitation of WKY and SHR neuronal cell lysates as described in "Methods." Immunoprecipitates containing 30 fmol/mg 125I-[Sar1-Ile8]-Ang II binding activities of AT1 receptors from WKY and SHR brain neurons were incubated with 0.3 IU MAP kinase at 30°C for the indicated time periods to determine the incorporation of 32P into the AT1 receptor essentially as described in "Methods." MBP (3 µg) was used as an assay control. Data are presented as picomoles of radioactivity incorporated into the AT1 receptor immunoprecipitate of WKY () or SHR () brain neurons or in MBP (•) as a function of time, and each point represents the mean±SE for three separate experiments. B and C, Immunoblot analysis of AT1 receptors phosphorylated in vitro by MAP kinase. AT1 receptors were subjected to MAP kinase–mediated phosphorylation essentially as described for A. Phosphorylated receptor preparations were electrophoresed in 10% SDS–polyacrylamide gel. Proteins were then transferred to membranes and subjected to autoradiography. Equal loading was determined by immunoblotting of AT1 receptors as evidenced by the bottom bands on the gel. B, Representative autoradiogram. C, Quantitation of the 32P-labeled band corresponding to the AT1 receptor, normalized for equal loading. Data are mean±SE (n=3).

View larger version (34K): [in this window] [in a new window]   Figure 4. Effect of the MAP kinase kinase inhibitor PD98059 on AT1 receptor phosphorylation in WKY and SHR neuronal cultures. Neuronal cultures were prelabeled with [32P]orthophosphate for 4 hours and incubated without (1 and 3) or with (2 and 4) 100 nmol/L Ang II for 10 minutes in the absence (1 and 2) or presence (3 and 4) of 10 µmol/L PD98059. Top, Representative autoradiogram. Bottom, Data (mean±SE, n=3), normalized for equal loading by immunoblotting of the membranes with AT1 receptor antibody essentially as described in the legend to Fig 1. *Significantly different from 1 (P<.05). #Significantly different from 2 (P<.05).

View larger version (24K): [in this window] [in a new window]   Figure 5. Effect of inhibitors of protein kinases on AT1 receptor phosphorylation in WKY and SHR neuronal cultures. Neuronal cultures were prelabeled with [32P]orthophosphate for 4 hours and incubated without (1, 3, 5, and 7) or with (2, 4, 6, and 8) 100 nmol/L Ang II for 10 minutes in the presence of 4 µmol/L lavendustin A (3 and 4), 1 µmol/L bis-indolylmaleimide (5 and 6), or 20 µmol/L H89 (7 and 8) at 37°C. Data (mean±SE, n=3) were normalized for equal loading by immunoblotting of the membranes with AT1 receptor antibody essentially as described in the legend to Fig 1. *Significantly different from 1 (P<.05).

Neuronal extracts were subjected to the immunoprecipitation protocol with the AT₁ receptor antibody followed by immunoblotting with MAP kinase antibody to determine whether the AT₁ receptor coprecipitates with MAP kinase. Fig 6 shows that a significant amount of MAP kinase appears to coimmunoprecipitate with AT₁ receptor protein in control neurons. Treatment of WKY brain neurons with 100 nmol/L Ang II resulted in a time-dependent increase in coimmunoprecipitation of AT₁ receptors with MAP kinase. The predominant band that coimmunoprecipitated with the AT₁ receptor was the p42 isoform. With Ang II, maximal coimmunoprecipitation approximately fourfold over control levels was observed in 5 minutes. A similar stimulation of coimmunoprecipitation of AT₁ receptor with MAP kinase was observed in Ang II–treated SHR brain neurons.

View larger version (24K): [in this window] [in a new window]   Figure 6. Coimmunoprecipitation of AT1 receptor with MAP kinase. WKY brain neuronal cultures were treated with 100 nmol/L Ang II for the indicated time periods. Cell-free lysates containing 400 µg protein were immunoprecipitated with AT1 receptor antibody and the immunoprecipitates (30 fmol/mg 125I-[Sar1-Ile8]-Ang II binding activity) subjected to SDS-PAGE. Separated proteins were immunoblotted with the use of MAP kinase antibody as described in "Methods." A, Representative autoradiograph. B, Quantitation of the protein band corresponding to MAP kinase. Data are mean±SE (n=3). *Significantly different from time zero (P<.05).

Finally, the effect of AT₁ receptor phosphorylation by MAP kinase on the binding of ¹²⁵I-[Sar¹-Ile⁸]-Ang II was measured to determine whether phosphorylated receptors retain Ang II binding activity. Fig 7 shows Scatchard analyses of competition-inhibition data for nonphosphorylated and phosphorylated AT₁ receptors from WKY and SHR brain neurons. B_max values for nonphosphorylated and phosphorylated receptors were 58±6 and 7±4 fmol/mg protein, respectively, for WKY brain neurons. The B_max values for nonphosphorylated and phosphorylated AT₁ receptor from SHR brain neurons were 83±7 and 9±5 fmol/mg protein, respectively. Thus, phosphorylation resulted in an approximate 89% decrease in the B_max in both WKY and SHR brain neurons. K_d values for nonphosphorylated receptors (0.5±0.03 nmol/L for both WKY and SHR) were comparable to those for MAP kinase–phosphorylated receptors (0.4±0.02 nmol/L for both WKY and SHR). These data show that MAP kinase–phosphorylated AT₁ receptors are much less able to bind ¹²⁵I-[Sar¹-Ile⁸]-Ang II in both WKY and SHR brain neurons.

View larger version (13K): [in this window] [in a new window]   Figure 7. Scatchard analysis of 125I-[Sar1-Ile8]-Ang II binding to MAP kinase–phosphorylated AT1 receptors in WKY and SHR brain neurons. Neuronal cell lysates were used to immunoprecipitate AT1 receptors from both strains of rats. Immunoprecipitates containing equal amounts of proteins (400 µg) were subjected to a phosphorylation protocol with exogenous MAP kinase essentially as described in "Methods." This procedure was followed by determining the ability of nonphosphorylated and phosphorylated AT1 receptors to bind 125I-[Sar1-Ile8]-Ang II as described in "Methods." A, indicates WKY nonphosphorylated; , WKY phosphorylated. B, • indicates SHR nonphosphorylated; , SHR phosphorylated. Each point represents triplicate samples.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
The observations presented in this study demonstrate that (1) Ang II stimulates phosphorylation of neuronal AT₁ receptors, (2) phosphorylation is comparable and proportional to the levels of AT₁ receptors in WKY and SHR brain neurons, (3) MAP kinase is a key protein kinase in this Ang II–induced AT₁ receptor phosphorylation, and (4) phosphorylated receptors lack ¹²⁵I-[Sar¹-Ile⁸]-Ang II binding activity.

Ang II–stimulated phosphorylation of neuronal AT₁ receptors is consistent with a similar observation in vascular smooth muscle cells.¹⁴ The site of this phosphorylation could be localized in the intracellular domain, consistent with the presence of threonine, serine, and tyrosine residues in this region of the receptor.^{20 21} Although the precise mechanism of this phosphorylation remains to be fully worked out, our data suggest a key role for MAP kinase: (1) Exogenous MAP kinase phosphorylates AT₁ receptors in both WKY and SHR brain neurons, (2) the MAP kinase kinase inhibitor PD98059 attenuates Ang II–stimulated phosphorylation (phosphorylation is not affected by inhibitors of protein tyrosine kinase or protein kinase A), (3) the MAP kinase coimmunoprecipitates with AT₁ receptor, and (4) the MAP kinase recognition sequence (amino acids^232-233) is present in the AT_1b receptor subtype. Both the AT_1a and AT_1b subtypes of AT₁ receptors are present in relatively equal proportions in WKY and SHR brain neuronal cells.⁵ These observations provide strong support for a direct involvement of MAP kinase in Ang II–induced phosphorylation of AT₁ receptors. However, alternate possibilities should not be totally discounted at the present. For example, the involvement of another GRK (such as ßARK₁) in the phosphorylation of AT₁ receptor cannot be ruled out. It is likely that Ang II stimulation of MAP kinase results in the phosphorylation and activation of other GRKs, which in turn phosphorylate the AT₁ receptor. This view is supported by recent data demonstrating AT₁ receptor phosphorylation by ßARK₁.²² In addition, the MAP kinase recognition sequence is present in ßARK₁.²³

An important question that arises from these studies concerns the physiological implications of AT₁ receptor phosphorylation. It is reasonable to suggest that the phosphorylation event may be responsible for Ang II– induced desensitization and internalization of AT₁ receptors from the neuronal plasma membrane. This view is supported by our data on the loss of ¹²⁵I-[Sar¹-Ile⁸]-Ang II binding ability of phosphorylated AT₁ receptors and consistent with the proposed role of phosphorylation in the agonist-induced desensitization of other GPCRs, such as ß- and -adrenergic receptors.^{10 11} Preliminary data indicating that Ang II induces internalization of cell-surface AT₁ receptors further support this proposal.²⁷

Neuronal cells from the SHR brain express twofold to fourfold higher functional AT₁ receptors than WKY brain neurons.⁵ This increase is associated with a 65% higher basal level of phosphorylated AT₁ receptor in these cells. However, the rate of phosphorylation, degree of stimulation by Ang II, and involvement of MAP kinase were comparable in the two strains of neurons. This observation indicates that the Ang II–mediated phosphorylation mechanism of the AT₁ receptor is similar in WKY and SHR brain neurons. In addition, phosphorylation results in a comparable loss of ¹²⁵I-[Sar¹-Ile⁸]-Ang II binding to AT₁ receptors in both strains of neurons. Thus, in spite of higher levels of AT₁ receptors and enhanced Ang II–induced neuromodulatory actions in SHR brain neurons,^{4 5} the rate of their inactivation is comparable, supporting our previous hypothesis that chronic neuromodulatory actions of Ang II on neurons may be distinctly regulated.

Ang II stimulates MAP kinase in neuronal cells in an Ras-Raf-1–dependent signaling pathway.⁸ This stimulation has been associated with Ang II regulation of chronic neuromodulation.^{8 9} Involvement of MAP kinase in AT₁ receptor-mediated regulation of neuromodulation is a unique event for a stimulatory GPCR that may involve interaction of one or more factors with the G_ß subunit of Gq.^{24 25} Thus, it is intriguing to suggest that activation of MAP kinase by Ang II potentially leads to both the stimulatory action of the AT₁ receptor on neuromodulation and desensitization followed by internalization of AT₁ receptors in neurons. A precise time sequence of signaling events will have to be elucidated and specific signaling molecules need to be identified to explain the specificity of these two opposing effects of Ang II mediated by AT₁ receptors.

	Selected Abbreviations and Acronyms

 

Ang II = angiotensin II ßARK1 = ß-adrenergic receptor kinase 1 AT1, AT2 = Ang II type 1, Ang II type 2 DMEM = Dulbecco’s modified Eagle’s medium ERK = extracellular signal-related kinase GPCR = G protein–coupled receptor GRK = G protein receptor kinase MAP = mitogen-activated protein MBP = myelin basic protein NET = norepinephrine transporter PAGE = polyacrylamide gel electrophoresis PDHS = plasma-derived horse serum PKC = protein kinase C SHR = spontaneously hypertensive rat(s) WKY = Wistar-Kyoto rat(s)

	Acknowledgments

 
This research was supported by grant HL-33610 from the National Institutes of Health, Bethesda, Md. We wish to thank Elizabeth Brown for cell-culture preparation, Jennifer Brock for the preparation of the manuscript, and Kevin Fortin for editorial suggestions.

Received September 19, 1996; first decision October 21, 1996; accepted February 5, 1997.

	References

Top Abstract Introduction Methods Results Discussion References

 
1. Yu K, Lu D, Rowland NE, Raizada MK. Angiotensin II regulation of tyrosine hydroxylase gene expression in the neuronal cultures of normotensive and spontaneously hypertensive rats. Endocrinology. 1996;137:2503-2513.[Abstract]

2. Lu D, Yu K, Paddy MR, Rowland NE, Raizada MK. Regulation of norepinephrine transport system by angiotensin II in neuronal cultures of normotensive and spontaneously hypertensive rat brains. Endocrinology. 1996;137:763-772.[Abstract]

3. Steckelings V, Lebrun C, Quadri F, Veltman A, Unger T. Role of brain angiotensin in cardiovascular regulation. J Cardiovasc Pharmacol. 1992;19(suppl 6):S73-S79.

4. Raizada MK, Lu D, Sumners C. AT₁ receptors and angiotensin actions in the brain and neuronal cultures of normotensive and hypertensive rats. In: Mukhopadhyay A, Raizada MK, eds. Current Concepts: Tissue Renin-Angiotensin System as Local Regulators in Reproductive and Endocrine Organs. New York, NY: Plenum Press; 1994:331-348.

5. Raizada MK, Lu D, Tang W, Kurian P, Sumners C. Increased angiotensin II type 1 receptor gene expression in neuronal cultures from spontaneously hypertensive rats. Endocrinology. 1993;132:1715-1722.[Abstract/Free Full Text]

6. Ding H, Zhou Q, Deng J, Lao HY, Yang K. Effect of overactivated central renin-angiotensin system on the concentration of brain norepinephrine and epinephrine in stroke-prone spontaneously hypertensive rats and its significance. Sheng Li Hsueh Pao. 1990;42:379-384.[Medline] [Order article via Infotrieve]

7. Yang K, Ding H, Zhou Q, Luo HY, Wu ZY. Central norepinephrine and angiotensin II contents in the brain regions of spontaneously hypertensive rats (SHR) and the interaction between them. Sheng Li Hsueh Pao. 1991;43:345-351.[Medline] [Order article via Infotrieve]

8. Yang H, Lu D, Yu K, Raizada MK. Regulation of neuromodulatory actions of angiotensin II in the brain neurons by the Ras-dependent mitogen-activated protein kinase pathway. J Neurosci. 1996;16:4047-4058.[Abstract/Free Full Text]

9. Lu D, Yang H, Raizada MK. Angiotensin II regulation of neuromodulation: downstream signalling mechanism from activation of mitogen-activated protein kinase. J Cell Biol. 1996,135:1609-1617.

10. Lefkowitz RJ. G-protein coupled receptor kinase. Cell. 1993;74:409-412.[Medline] [Order article via Infotrieve]

11. Premont RT, Inglese J, Lefkowitz RJ. Protein kinases that phosphorylate activated G-protein–coupled receptors. FASEB J. 1995;9:175-182.[Abstract]

12. Raizada MK, Muther TF, Sumners C. Increased angiotensin II specific receptors in neuronal culture of spontaneously hypertensive rat brain. Am J Physiol. 1984;247:C364-C372.[Medline] [Order article via Infotrieve]

13. Marrero MB, Schieffer B, Paxton WG, Heerdt L, Berk BC, Delafontaine P, Bernstein KE. Direct stimulation of Jak/STAT pathway by the angiotensin II AT₁ receptor. Nature. 1995;375:247-250.[Medline] [Order article via Infotrieve]

14. Paxton WG, Marrero MB, Klein JD, Delafontaine P, Berk BC, Bernstein KE. The angiotensin II AT₁ receptor is tyrosine and serine phosphorylated and can serve as a substrate for the src family of tyrosine kinases. Biochem Biophys Res Commun. 1994;200:260-267.[Medline] [Order article via Infotrieve]

15. Abramowski D, Staufenbiel M. Identification of the 5-hydroxytryptamine-2C receptor as a 60-kDa N-glycosylated protein in choroid plexus and hippocampus. J Neurochem. 1995;65:782-790.[Medline] [Order article via Infotrieve]

16. Paxton WG, Runge M, Horaist C, Cohen C, Alexander RW, Bernstein KE. Immunohistochemical localization of rat angiotensin II AT₁ receptor. Am J Physiol. 1993;264:F989-F995.[Medline] [Order article via Infotrieve]

17. Kato S, Endoh H, Masuhiro Y, Kitamoto T, Uchiyama S, Sasaki H, Masushige S, Gotoh Y, Nishida E, Kawashima H, Metzger D, Chambon P. Activation of the estrogen receptor through phosphorylation by mitogen-activated protein kinase. Science. 1995;270:1491-1494.[Abstract/Free Full Text]

18. Morrison P, Takishima K, Rosner MR. Role of threonine residues in regulation of the epidermal growth factor receptor by protein kinase C and mitogen-activated protein kinase. J Biol Chem. 1993;268:15536-15543.[Abstract/Free Full Text]

19. Alessi DR, Cuenda A, Cohen P, Dudley DT, Saltie AR. PD98059 is a specific inhibitor of the activation of mitogen-activated protein kinase kinase in vitro and in vivo. J Biol Chem. 1995;270:27489-27494.[Abstract/Free Full Text]

20. Inagami T, Iwai N, Sasaki K, Yamano Y, Bardhan S, Chaki S, Guo DF, Furuta H. Cloning, expression and regulation of angiotensin II receptors. In: Raizada MK, Phillips MI, Sumners C, eds. Cellular and Molecular Biology of Renin-Angiotensin System. Boca Raton, Fla: CRC Press; 1993:273-291.

21. Catt KJ, Sandberg K, Balla T. Angiotensin II receptor and signal transduction mechanisms. In: Raizada MK, Phillips MI, Sumners C, eds. Cellular and Molecular Biology of the Renin-Angiotensin System. Boca Raton, Fla: CRC Press; 1993:307-356.

22. Oppermann M, Freedman NJ, Alexander RW, Lefkowitz RJ. Phosphorylation of the type 1A angiotensin II receptor by G-protein– coupled receptor kinases and protein kinase C. J Biol Chem. 1996;271:13266-13272.[Abstract/Free Full Text]

23. Arriza JL, Dawson TM, Simerly RB, Martin LJ, Caron MG, Snyder SH, Lefkowitz RJ. The G-protein–coupled receptor kinases ß-ARK₁ and ß-ARK₂ are widely distributed at synapses in rat brain. J Neurosci. 1992;12:4045-4055.[Abstract]

24. Hawes BE, VanBiesen TV, Koch WJ, Luttrell LM, Lefkowitz RJ. Distinct pathways of Gi- and Gq-mediated mitogen-activated protein kinase activation. J Biol Chem. 1995;270:17148-17153.[Abstract/Free Full Text]

25. Inglese J, Koch WJ, Touhara K, Lefkowitz RJ. G_ß interactions with PH domains and Ras-MAPK signaling pathways. Trends Biochem Sci. 1995;20:151-156.[Medline] [Order article via Infotrieve]

26. Yang H, Lu D, Raizada MK. Lack of cross-talk between ₁-adrenergic and AT₁ receptors in neurons of spontaneously hypertensive rat brain. Hypertension. 1996;27:1277-1283.[Abstract/Free Full Text]

27. Yang H, Lu D, Vinson GP, Raizada MK. Involvement of MAP kinase in angiotensin II–induced phosphorylation and intracellular targeting of neuronal AT₁ receptors. J Neurosci. 1997;17:1660-1669.[Abstract/Free Full Text]

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