ANG II type 1 receptor downregulation does not require receptor endocytosis or G protein coupling

J. Gregory Modrall1, Masakatsu Nanamori2, Junichi Sadoshima3, Douglas C. Barnhart4, James C. Stanley4, and Richard R. Neubig2,3

1 Department of Surgery, Dallas Veterans Affairs Medical Center and the University of Texas Southwestern Medical Center, Dallas, Texas 75235; and Departments of 4 Surgery, 2 Pharmacology, and 3 Medicine, University of Michigan Medical Center, Ann Arbor, Michigan 48109


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

ANG II type 1 (AT1) receptors respond to sustained exposure to ANG II by undergoing downregulation of absolute receptor numbers. It has been assumed previously that downregulation involves endocytosis. The present study hypothesized that AT1 receptor downregulation occurs independently of receptor endocytosis or G protein coupling. Mutant AT1 receptors with carboxy-terminal deletions internalized <5% of radioligand compared with 65% for wild-type AT1 receptors. The truncated AT1 receptors retained the ability to undergo downregulation. These data suggest the existence of an alternative pathway to AT1 receptor degradation that does not require endocytosis, per se. Point mutations in either the second transmembrane region or second intracellular loop impaired G protein (Gq) coupling. These receptors exhibited a biphasic pattern of downregulation. The earliest phase of downregulation (0-2 h) was independent of coupling to Gq, but no additional downregulation was observed after 2 h of ANG II exposure in the receptors with impaired coupling to Gq. These data suggest that coupling to Gq is required for the later phase (2-24 h) of AT1 receptor downregulation.

internalization; clathrin-coated pit; radioligand binding assay


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

INTERACTION OF ANG II with the ANG II type 1 (AT1) receptor induces vasoconstriction, sodium reabsorption, and stimulation of aldosterone release (19). In addition, activation of the AT1 receptor modulates growth of vascular smooth muscle cells (VSMCs) and cardiac myocytes (15). Pathological activation of the AT1 receptor has been implicated in hypertension, vascular remodeling, nephrosclerosis, and cardiac hypertrophy (15, 20). Because AT1 receptor blockade may ameliorate these conditions, it is likely that cell surface expression of the AT1 receptor is a key determinant of whether homeostasis is maintained or pathological changes ensue.

The stereotypical response of the AT1 receptor to prolonged exposure to its ligand, ANG II, is to undergo one or more of the following three processes to blunt the subsequent physiological response: desensitization, endocytosis, or downregulation (5, 11, 13, 17, 23, 24, 28). Desensitization appears to result from phosphorylation of the AT1 receptor by two kinases (23), uncoupling the receptor from its G protein and downstream signaling pathways. With continued exposure to ANG II, the AT1 receptor undergoes endocytosis, physically removing receptors from the cell surface (5, 11, 13, 17, 24, 28). Chronic exposure to increased levels of ANG II results in downregulation of total receptor numbers (17, 24).

Downregulation of the AT1 receptor is poorly understood but probably involves both decreased production of receptors and increased clearance and degradation of cell surface receptors (1, 24). The present investigation focused on the latter process. Traditionally, increased receptor degradation has been assumed to involve or require receptor endocytosis (24). To better understand the process of AT1 receptor downregulation, we tested this assumption by hypothesizing that AT1 receptor downregulation occurs independently of receptor endocytosis. In addition, we sought to investigate whether G protein coupling is necessary for AT1 receptor downregulation by challenging the null hypothesis that AT1 receptor downregulation does not require G protein coupling.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Materials

The full-length rat AT1 receptor cDNA in a pCDM8 eukaryotic expression vector was kindly provided by Dr. Jeff Harrison. The eukaryotic expression vector pCDM8 and Escherichia coli MC1061/P were from Invitrogen (Carlsbad, CA). The Quick Change site-directed mutagenesis kit and XL1-Blue E. coli were from Stratagene (La Jolla, CA). Restriction enzymes, DNA ligase, PCR reagents, and Taq polymerase were from Promega (Madison, WI). Plasmid Midi Kit for plasmid purification and QIAquick gel extraction kit were from Qiagen (Valencia, CA). COS-7 cells were supplied by Dr. John Lowe, University of Michigan. DMEM, low-serum MEM (Opti-MEM), FBS, Lipofectamine reagent, and PBS were from BRL Life Technologies (Rockville, MD). High-glucose DMEM was from Irvine Scientific (Santa Ana, CA). 125I-labeled ANG II (2,200 Ci/mmol) was from Dupont-NEN (Boston, MA). myo-[3H]inositol (73-112 Ci/mmol) was from Amersham (Piscataway, NJ). DC Protein Assay kit, 10% polyacrylamide gels, and Dowex AG1-X8 anion exchange resin were from Bio-Rad (Hercules, CA). Nitrocellulose membranes were from Schleicher and Schuell (Keene, NH). Rabbit anti-AT1 receptor primary antibody was from Research Diagnostics (Flanders, NJ). Goat anti-rabbit secondary antibody was from Santa Cruz Biotechnology (Santa Cruz, CA). The enhanced chemiluminescence (ECL) kit was from Pierce (Rockford, IL). Scintiverse was from Fisher Scientific (Houston, TX). Fura 2-AM was from Molecular Probes (Eugene, OR). BSA (fraction V, >= 98% purity), glutamine, antibiotics, trypsin, ANG II, saralasin, and chemicals were from Sigma (St. Louis, MO). All chemicals were reagent grade or better.

Receptor Mutagenesis

The full-length rat AT1 receptor in a pCDM8 eukaryotic expression vector served as the source of wild-type receptor in all transfection experiments (29). To determine if endocytosis is required for AT1 receptor downregulation, endocytosis-deficient mutants were constructed. It was shown previously that the carboxy terminus of the AT1 receptor is critical for receptor endocytosis (5, 22, 28). Accordingly, two carboxy-terminal truncations of the AT1 receptor were constructed by introducing a stop codon at amino acid 310 or 312 of the wild-type receptor using PCR, which resulted in a mutant receptor missing the terminal 49 or 47 amino acids, respectively (5, 28). These mutants were designated AT1-309T and AT1-311T. Two endocytosis-deficient mutants were constructed because it was believed that the AT1-309T and AT1-311T mutants would differ phenotypically in their ability to couple to G proteins (Gq; see Refs. 13, 22, 27). The PCR primers used to construct the AT1-309T mutant were as follows: sense, 5'-CCACTGCTTAC TGGCTTATC-3'; antisense, 5'-TGCTCTAGAACTAAAATTTCTTCCCCAGAAAGCCGTA GAACAGAGG-3' (mutations underlined). The PCR primers used to create the AT1-311T construct were as follows: sense, 5'-CATGATCAAGCTTGGTACC GAGCTCGGA-3'; antisense, 5'-CGCTCTAGACGTCACTTTTTAAATTTCTTCCCCAG-3' (mutations underlined). Primers included a 5' HindIII site and a 3' XbaI site flanking the cDNA insert. After the desired PCR product was resolved on a DNA gel and purified by gel extraction, the resulting 1.5-kb fragments were digested with HindIII and XbaI, gel purified, and ligated into the complementary sites of the pCDM8 expression vector. Each construct was used to transform E. coli MC1061/P. After antibiotic selection and amplification of the transformed E. coli, plasmid DNA was purified using a kit from Qiagen. Constructs were confirmed by dideoxy sequencing of amplified cDNA inserts.

To determine if G protein coupling is necessary for downregulation, mutants with reduced ability to activate phospholipase C were constructed. Based on the mutagenesis studies of Shibata et al. (27) and Ohyama et al. (22), a series of point mutations were introduced in the conserved DRY motif in the second intracellular loop (i2 LOOP) of the wild-type AT1 receptor. Asp125, Arg126, Tyr127, and Met134 were converted to Gly125, Gly126, Ala127, and Ala134, respectively. This construct was designated the AT1-i2 LOOP mutant. These mutations were introduced using overlap extension PCR (9). The 5'-fragment was prepared using the following primers: sense, 5'-CCACTGCTTACTGGCTTATC-3'; antisense, 5'-ATGGCCAGGGCGCCG CCGATGCTGAGACACGTGAGAAAG-3' (mutations underlined). The 3'-fragment was created with the following primers: sense, 5'-TTCTCACGTGTCTCAGCATCGGCGGCGCCCTGG CCATCGTCCACCCAGCGAAGTCTCGCCTTCGCCGCACGATGC-3' (mutations underlined); antisense, 5'-AGCAGTAG CCTCATCACAC-3'. The primers for full-length amplification were sense, 5'-CCATGCTTACTGGCTTATC-3' and antisense, 5'-AGCAGTAGCCTCATCATCAC-3'. After HindIII and XbaI digestion, the full-length product was subcloned into the pCDM8 vector. The plasmid DNA was then amplified, purified, and sequenced as above.

An additional mutant with impaired G protein coupling was constructed by modifying the second transmembrane region of the wild-type AT1 receptor. On the basis of the work of Hunyady et al. (13), Asp74 was changed to Asn74 using the Quick Change site-directed mutagenesis kit. This construct was termed the AT1-D74N mutant. The PCR primers used were sense, 5'-AATCT CGCCTTGGCTAATTTATGCTTTTTGCTG-3' and antisense, 5'-CAGCAAAAAGCATAAATTAGCCAAGGCGAGATT-3' (mutations underlined). The construct was transformed into XL1-Blue E. coli. The plasmid DNA was then amplified, purified, and sequenced as above.

Cell Culture and Transfection

COS-7 cells were maintained in a humidified 5% CO2 atmosphere at 37°C in high-glucose DMEM supplemented with 10% FBS, 20 mM glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin. COS-7 cells were seeded on 100-mm plates 24 h before transfection.

At 60-80% confluence, COS-7 cells in 100-mm plates were rinsed with serum- and antibiotic-free DMEM, and transfections were performed. Briefly, plasmid DNA was mixed with 6 µl of Lipofectamine/1 µg of DNA for 45 min in Opti-MEM at room temperature. The mixture was then diluted to a final volume of 9 ml with Opti-MEM, added to cells in 100-mm plates, and swirled gently. The cells and DNA were incubated for 24 h under standard culture conditions. COS-7 cells were then rinsed with DMEM and subcultured into 24-well plates for binding assays, 100-mm plates for phosphoinositide assays, or coverslips in 30-mm plates for calcium assays. Cells were cultured an additional 48 h with standard culture conditions before assays.

Receptor Binding Assays

Saturation binding curves. Saturation binding curves were determined using a modification of whole cell receptor binding assays (30). Briefly, 48 h after transfection, each well was aspirated and rinsed with assay buffer (Opti-MEM, 0.1% BSA). Assay buffer containing 30 pM 125I-ANG II and varying concentrations of unlabeled ANG II (0-300 nM) was added to cells and incubated in a volume of 0.3 ml for 1 h at room temperature. Binding was terminated by aspirating the medium and gently rinsing cells with ice-cold Opti-MEM. Cells were solubilized with 0.5 M NaOH-0.05% SDS, and total radioligand bound was quantitated by gamma counting. Nonspecific binding (<5% of total binding) was determined by adding 1 µM unlabeled ANG II or saralasin. Protein assay was performed on each sample using the Bio-Rad DC kit, which is a modification of the method of Lowry et al. (18). Receptor density was expressed as femtomoles of receptor per milligram of protein. Maximal binding (Bmax) and dissociation constant (Kd) values for saturation binding curves were determined by nonlinear least-squares analysis using a single hyperbolic binding function (Graphpad Prism).

Endocytosis. Endocytosis of the AT1 receptor was defined as the fraction of bound 125I-ANG II that became acid resistant in whole cell binding assays (30). Endocytosis studies were performed in triplicate at 37°C. Each well was aspirated and rinsed with assay buffer at 37°C before addition of radioligand. 125I-ANG II (0.5 nM) in a volume of 0.3 ml was added to cells to initiate binding. After incubation for the prescribed time at 37°C, binding was terminated by rapid removal of the incubation medium and gentle rinsing three times with ice-cold Opti-MEM. Surface-bound 125I-ANG II was removed using the acid wash technique of Crozat et al. (7) in which cells were exposed to 50 mM glycine-150 mM NaCl, pH 3, for 10 min at 4°C. Surface-bound radioactivity was collected by careful aspiration of the acid wash solution and three rinses with ice-cold Opti-MEM. The cell-associated radioactivity was measured by harvesting cells, and both fractions were quantitated by gamma counting. Nonspecific binding was determined by treating cells transfected with the cDNA of interest with an excess of unlabeled ANG II in the presence of 125I-ANG II (0.5 nM) and accounted for <5% of total binding. Percent endocytosis was calculated from the fraction of intracellular (cell-associated) radioactivity as a percentage of total binding (cell surface plus cell-associated radioactivity).

The validity of the acid wash technique was confirmed in pilot experiments. Cells were incubated with assay buffer containing 125I-ANG II at 4°C to prevent endocytosis. Cells were subjected to acid wash and rinsed three times. The amount of residual cell-associated radioactivity was reduced to the level of nonspecific binding. Subsequent incubation with 125I-ANG II restored surface-bound radioactivity to preacid wash levels, confirming the functional integrity of binding sites after the acid wash (data not shown).

Downregulation. Downregulation of the AT1 receptor was quantitated using whole cell receptor binding assays as described above with the following modifications. At intervals before binding assays, the medium was aspirated, and cells were pretreated with 100 nM ANG II or vehicle in assay buffer at 37°C for the prescribed time. In all experiments, pretreatment with ANG II or vehicle was initiated at least 48 h after transfection and was completed 72 h after transfection. Pretreatment was started at variable times before the binding assay and was terminated simultaneously to permit quantitation of AT1 receptor density in parallel for all transfected cells after pretreatment. After pretreatment with ANG II, cells were rinsed and subjected to a standard acid wash at 4°C to remove surface-bound unlabeled ANG II. After rinsing three times, the binding assay was performed using 0.3 ml assay buffer containing 0.1 nM 125I-ANG II for 1 h at room temperature. Ullian and Linas (30) noted that AT1 receptors recycle rapidly (tau 1/2approx 15 min) to the cell surface at room temperature in the absence of excessive ANG II. Hence, a binding assay of 1 h duration at room temperature quantitated all remaining receptors, internalized or not. Total and nonspecific binding was determined as described above. Percent downregulation was calculated by expressing specific binding (fmol receptor/mg protein) of ANG II-treated cells as a percentage of specific binding of control cells. Control cells were transfected with the cDNA of interest and were treated in parallel with vehicle, rather than ANG II. Each construct served as its own control.

Immunoblotting

At the termination of downregulation experiments, transfected COS-7 cells were washed with PBS and lysed at 4°C for 15 min in 1 mM Tris base, pH 7.4, containing protease inhibitors (10 mM benzamidine, 10 U/ml aprotinin, and 1 mM phenylmethylsulfonyl fluoride). Cells were scraped, homogenized, and centrifuged (1,000 g, 10 min) at 4°C to remove nuclei and undisrupted cells. The pellet was homogenized and centrifuged again two times, as above. Supernatants from each spin were combined and centrifuged (40,000 g, 30 min). The pellet was resuspended in 50 mM Tris, 10 mM MgCl2, and 1 mM EGTA (pH 7.6) and stored at -70°C. Protein assay was performed on each sample using the Bio-Rad DC kit as before. Cellular protein (20 µg) in a total volume of 20 µl, including loading buffer (0.38 Tris base, 8% SDS, 4 mM EDTA, and 10 mg/ml bromphenol blue), was boiled for 5 min and loaded on 10% polyacrylamide gels for gel electrophoresis. Negative control (running buffer alone) and molecular weight markers were run in parallel. Gels were transferred to nitrocellulose membranes, washed with Tris-buffered saline, and blocked with blotto for 2-4 h at 4°C. Rabbit anti-AT1 receptor primary antibody (1:200 dilution), diluted in blotto, was added overnight at 4°C. The next day, membranes were washed and reacted with goat anti-rabbit secondary antibody for 2 h while shaking. After being rinsed, reagents for ECL were added according to the manufacturer's (Pierce) instructions. Membranes were exposed to X-ray film. Signal intensity was quantitated with a densitometer.

Phosphoinositide Production

Measurement of inositol phosphates (IPx) was based on the method of Berridge et al. (3), as described by Thompson et al. (29). After transfection (24 h), cells were incubated with myo-[3H]inositol (10 µCi/ml) in DMEM for 24 h at 37°C. Labeling was terminated by aspirating the medium, rinsing cells with oxygenated reaction buffer (142 mM NaCl, 30 mM HEPES buffer, pH 7.4, 5.6 mM KCl, 3.6 mM NaHCO3, 2.2 mM CaCl2, 1.0 mM MgCl2, and 1 mg/ml D-glucose), and harvesting cells with PBS-0.02% EDTA. Cells were centrifuged two times (300 g, 5 min) in reaction buffer, and the pellet was resuspended in an equal volume of reaction buffer containing 60 mM LiCl (final concentration 30 mM LiCl). Inositol phosphate production was stimulated by mixing 0.25 ml of cell suspension with 0.25 ml of 0-100 nM ANG II in reaction buffer (without LiCl). The mixture was incubated for 30 min at 37°C, and then ice-cold 0.5 ml 20% TCA was added. Precipitates were pelleted (4,100 g, 20 min), and the TCA-soluble fraction was aspirated, washed with water-saturated diethyl ether, and neutralized with NaHCO3. IPx were isolated by adsorption to 0.5 ml Dowex AG1-X8 formate resin slurry and rinsed five times with 3 ml unlabeled 5 mM myo-inositol, followed by elution with 1 ml of 1.2 M ammonium formate-0.1 M formic acid. The eluates were counted by liquid scintillation spectroscopy in 5 ml Scintiverse.

To normalize released IPx for lipid content, the TCA-insoluble fraction of each sample was processed for measurement of lipids. After resuspending the pellet in 0.5 ml H2O and 1.5 ml chloroform-methanol (1:2), a 200-µl aliquot of the organic phase was counted by liquid scintillation spectroscopy in 5 ml of Scintiverse.

Calcium Measurements

Calcium measurements were performed as described previously (2). Cells were harvested 24 h after transfection and plated on polylysine-coated glass coverslips at a density of 2 × 105 cells in 200 µl of DMEM. After permitting cells to adhere to coverslips for 4 h, 1.5 ml of DMEM was added, and cells were incubated for 24 h at 37°C in a humidified 5% CO2 environment. Cells were loaded with fresh warmed 1 µM fura 2-AM in 0.5% DMSO for 30 min at 37°C. Coverslips were then washed, rinsed in control buffer (118 mM NaCl, 4.7 mM KCl, 1.8 mM CaCl2, 10 mM HEPES, 15 mM NaHCO3, 11 mM glucose, 0.9 mM NaH2PO4, 0.8 mM MgSO4, and 0.1% BSA, pH 7.4), and placed in a Lucite superfusion chamber of 1 ml volume. The superfusion rate of buffer with or without 100 nM ANG II was 1 ml/min at 37°C.

Single-cell intracellular calcium concentration ([Ca2+]i) measurements were performed using a Zeiss Axiovert inverted microscope and Attofluor digital imaging system. [Ca2+]i was calculated from the ratios of the fluorescence intensities of fura 2-AM at excitation wavelengths of 334 and 380 nm with an emission wavelength of 540 nm. Calibration of the system was performed as described previously (2). A single representative microscope field containing ~50-70 cells/coverslip was chosen for examination. To exclude damaged cells, only cells displaying basal [Ca2+]i levels of <150 nM were included for study. A cell was judged to have responded to ANG II if its [Ca2+]i increased by >50 nM over baseline.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The wild-type AT1 receptor transiently expressed in COS-7 cells displayed high-affinity binding of ANG II (Kd = 1.2 nM) comparable to VSMCs (17). The mutant constructs AT1-309T, AT1-311T, AT1-i2 LOOP, and AT1-D74N also displayed receptor affinities in COS-7 cells ranging from 2.4 to 8.6 nM (Table 1). Expression of mutant receptors ranged from ~2 to 10 times that of wild-type AT1 receptor (Table 1). Calculation of the Hill slope from competition binding curves confirmed that each of the AT1 receptor constructs has a single class of AT1 receptors (data not shown).

                              
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Table 1.   Binding parameters

To determine if endocytosis is required for AT1 receptor downregulation, several approaches were used to attempt to inhibit endocytosis. Potassium depletion and treatment with hyperosmolar (sucrose) medium have been used to inhibit endocytosis with brief exposures (12, 16) but were visibly toxic to COS-7 cells during longer exposures (>= 4 h). Phenylarsine oxide has also been used to block AT1 receptor endocytosis (17) but provided only modest inhibition of endocytosis (<50% of endocytosis inhibited) in our pilot studies (data not shown). Because of the shortcomings of these approaches, a receptor mutagenesis strategy was employed to impair endocytosis.

Prior studies demonstrated that the carboxy-terminus of the wild-type AT1 receptor is critical for endocytosis (5, 28), so a premature stop codon was introduced at amino acid 310 or 312 of the wild-type AT1 receptor to create the endocytosis-deficient mutants AT1-309T and AT1-311T, respectively. Loss of normal endocytosis was confirmed by comparing these mutants with the wild-type AT1 receptor in their ability to undergo endocytosis of tracer 125I-ANG II at 37°C (Fig. 1). Percent endocytosis was calculated by determining the percentage of specific binding that remained cell associated after acid washing. Wild-type AT1 receptor demonstrated a maximum of ~65% endocytosis at 60 min, with the majority (52%) occurring in the initial 15 min. In contrast, the AT1-309T and AT1-311T mutants demonstrated minimal endocytosis, reaching 5% endocytosis at 60 min. Thus the deletion mutants were truly "endocytosis deficient."


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Fig. 1.   Endocytosis of wild-type and deletion mutation ANG II type 1 (AT1) receptors. Endocytosis of 125I-labeled ANG II at 37°C by wild-type AT1 and AT1-309T and AT1-311T mutant receptors was quantitated as described in MATERIALS AND METHODS. Data are expressed as percentage of total binding that is cell associated (internalized) at each time point and are means ± SE for 3 independent experiments, each performed in duplicate.

To determine if endocytosis is required for AT1 receptor downregulation, the wild-type AT1 receptor and endocytosis-deficient mutants were subsequently compared in their ability to undergo downregulation in response to treatment with 100 nM ANG II (Fig. 2). After stripping receptor-bound, unlabeled ligand by acid washing, a standard 1-h binding assay was used to quantitate receptor density. Downregulation was quantitated by expressing receptor density as a percentage of that in vehicle-treated control cells. The wild-type AT1 receptor exhibited a rapid loss of binding sites when exposed to 100 nM ANG II. Approximately 35% of wild-type AT1 binding sites were lost in the initial 4 h of ANG II exposure. A second, slower phase of downregulation claimed an additional 15% of wild-type AT1 binding sites over the ensuing 16 h, yielding a total of 50% downregulation at 24 h of ANG II treatment. This plateau of wild-type AT1 downregulation continued for 48 h of ANG II exposure (data not shown). The endocytosis-deficient mutants AT1-309T and AT1-311T demonstrated an initial phase of downregulation (0-8 h) that lagged slightly relative to the wild-type AT1 receptor. During the ensuing 16 h of ANG II exposure, the endocytosis-deficient mutants were essentially indistinguishable from the wild-type AT1 receptor in their ability to undergo downregulation in response to ANG II. Adjusting expression of wild-type and endocytosis-deficient mutants to achieve equivalent Bmax (0.3 ± 0.1 vs. 0.5 ± 0.1 fmol/mg protein for wild-type AT1 receptor and AT1-311T, respectively) did not alter the patterns of downregulation depicted in Fig. 2. In addition, immunoblotting of COS-7 cell lysates for the AT1 receptor confirmed downregulation of total receptor number for both wild-type AT1 and endocytosis-deficient mutant receptors after treatment with 6 h of 100 nM ANG II (Fig. 3). Taken together, these data suggest that minimal, if any, endocytosis appears necessary for AT1 receptor downregulation.


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Fig. 2.   Downregulation of wild-type and endocytosis-deficient mutant AT1 receptor binding sites. AT1 receptor downregulation by wild-type AT1 and AT1-309T and AT1-311T mutant receptors in response to exposure to 100 nM ANG II at 37°C. Receptor density was quantitated by radioligand binding assay, as described in MATERIALS AND METHODS. Data are expressed as a percentage of control binding at each time point and are means ± SE for 4 independent experiments, each performed in duplicate.



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Fig. 3.   Downregulation of wild-type and endocytosis-deficient mutant AT1 receptor protein. Wild-type AT1 and AT1-311T mutant receptors were transiently expressed in COS-7 cells and exposed to either vehicle or 100 nM ANG II for 6 h at 37°C. Receptor protein was quantitated by immunoblotting of cell lysates with anti-AT1 antibody, as described in MATERIALS AND METHODS. A representative blot from 3 independent experiments is presented.

The data above cannot exclude the possibility that normal receptor endocytosis may be occurring, but as a result of rapid degradation of radioligand-receptor complexes, minimal accumulation of cell-associated radioactivity is seen. To eliminate this possibility, a variation of the standard endocytosis assay was performed. COS-7 cells transiently transfected with either wild-type AT1 receptor or AT1-309T were pretreated with 125I-ANG II at 4°C for 4 h to permit binding of radioligand to receptors without endocytosis. After removing unbound radioligand, cells were incubated at 37°C in wash buffer containing unlabeled ANG II in excess (1 µM). The percentage of total 125I-ANG II that remained at the cell surface vs. that percent which became cell associated (internalized) or translocated to the medium was quantitated to determine the rate of loss of surface receptors. Wild-type AT1 receptor demonstrated rapid loss of cell surface radioligand and rapid accumulation of intracellular (cell-associated) radioligand, consistent with its ability to undergo rapid endocytosis (Fig. 4A). By comparison, the AT1-309T mutant lost cell surface radioactivity at a much slower rate with minimal intracellular accumulation of radioligand (Fig. 4B). In fact, most of the loss of cell surface radioactivity from AT1-309T mutants appeared in the medium, which was probably a result of dissociation of cell surface 125I-ANG II and replacement by unlabeled ANG II. Taken together, these data exclude the possibility of rapid receptor endocytosis and degradation.


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Fig. 4.   Loss of cell surface 125I-ANG II from wild-type and deletion mutation AT1 receptors. Wild-type AT1 receptor (A) or AT1-309T receptor (B) was transiently expressed in COS-7 cells. Transfected cells were prelabeled with 125I-ANG II at 4°C for 4 h to permit equilibrium binding with minimal endocytosis. After removing unbound radioligand, the experiment was initiated by the addition of unlabeled ANG II in excess (1 µM) at 37°C. The percentage of total 125I-ANG II that remained at the cell surface vs. the percentage that became cell associated (intracellular) or translocated to the medium was quantitated to determine the rate of loss of surface receptors. Data are expressed as percentage of total binding at each time point. Data are representative of 2 independent experiments.

Although it is recognized that AT1 receptor endocytosis does not require G protein coupling (6, 13), it is not known if G protein coupling is necessary for downregulation of the AT1 receptor. This issue was addressed by constructing mutant AT1 receptors with impaired coupling to Gq and determining their ability to undergo downregulation. It was reported previously that mutations of the i2 LOOP and second transmembrane domain of the wild-type AT1 receptor impair Gq-mediated responses (13, 22, 27). These mutations were introduced, as described in MATERIALS AND METHODS, to create the AT1-i2 LOOP and AT1-D74N constructs, respectively. The binding characteristics of these mutants are summarized in Table 1. Next, uncoupling of these constructs from Gq was confirmed. The AT1-i2 LOOP mutant did not display ANG II-stimulated phosphoinositide turnover (Fig. 5 and Table 2) or calcium mobilization (data not shown), suggesting complete uncoupling from Gq. The AT1-D74N construct demonstrated impaired agonist-stimulated phosphoinositide turnover (Fig. 5 and Table 2) and calcium mobilization (data not shown), but it clearly was able to couple to Gq.


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Fig. 5.   ANG II-stimulated phosphoinositide release. Wild-type AT1, AT1-i2 LOOP, and AT1-D74N mutant receptors or pCDM8 vector alone were transiently expressed in COS-7 cells. Inositol phosphates (inositol monophosphate, inositol bisphosphate, and inositol trisphosphate, or IPx) were measured as described in MATERIALS AND METHODS. Data are presented as means ± SE for 3 independent experiments, each performed in triplicate. Brackets denote concentration.


                              
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Table 2.   IPX release

The impact of uncoupling from Gq was assessed with standard downregulation experiments. Upon exposure to 100 nM ANG II, the pattern of downregulation observed for the AT1-i2 LOOP and AT1-D74N constructs was biphasic (Fig. 6). During the earliest phase (0-2 h) of downregulation, the AT1-D74N mutant paralleled the wild-type AT1 receptor. During the subsequent phase of downregulation (2-24 h), the AT1-D74N receptor recovered ANG II binding sites despite ongoing ANG II exposure. These data cannot distinguish whether the recovery of binding sites by the AT1-D74N receptor represents recycling of receptors vs. synthesis of new receptors. In any case, receptor density during this phase of downregulation approached that of untreated control cells. The AT1-i2 LOOP mutant demonstrated a similar biphasic pattern of downregulation that was intermediate between the wild-type AT1 and AT1-D74N constructs. The AT1-i2 LOOP construct paralleled the wild-type AT1 receptor during the initial 4 h of downregulation but recovered a smaller fraction of its AT1 binding sites during the subsequent 16 h of ANG II treatment. The AT1-i2 LOOP construct plateaued at a receptor density equivalent to 80% of control cells. Thus the initial phase of downregulation of the AT1-i2 LOOP and AT1-D74N mutants was indistinguishable from the wild-type AT1 receptor, so it appears that coupling to Gq is not necessary for this phase of AT1 receptor downregulation. During the apparent second phase of downregulation, there appears to be no additional downregulation of either AT1-i2 LOOP or AT1-D74N mutants beyond that achieved during the initial phase of downregulation. This observation seems to indicate that coupling to Gq may be critical for the second phase of downregulation.


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Fig. 6.   Downregulation of wild-type AT1 and mutant AT1 receptors with impaired G protein coupling. AT1 receptor downregulation by wild-type AT1, AT1-i2 LOOP, and AT1-D74N mutant receptors in response to exposure to 100 nM ANG II at 37°C was quantitated as described in MATERIALS AND METHODS. Data are expressed as percentage of control binding at each time point and are means ± SE for 4 independent experiments, each performed in duplicate.

The AT1-i2 LOOP and AT1-D74N mutants also provided further evidence to dissociate AT1 receptor endocytosis and downregulation. These mutants retained the ability to undergo endocytosis normally (Fig. 7) but demonstrated impaired downregulation (Fig. 6). Together with the prior data, these data suggest that receptor endocytosis is neither necessary nor sufficient for downregulation of the AT1 receptor.


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Fig. 7.   Endocytosis of wild-type AT1 and mutant AT1 receptors with impaired G protein coupling. Endocytosis of 125I-ANG II at 37°C by wild-type AT1 and AT1-i2 LOOP and AT1-D74N mutant receptors was quantitated as described in MATERIALS AND METHODS. Data are expressed as percentage of total binding that is cell associated (internalized) at each time point and are means ± SE for 3 independent experiments, each performed in duplicate.

To rule out coupling to Gi as a contributor to AT1 receptor downregulation, the wild-type AT1 receptor was pretreated with 100 ng/ml pertussis toxin for 16 h before initiating downregulation experiments. This dose of pertussis toxin had previously been demonstrated to block Gi-mediated inhibition of adenylate cyclase activity by alpha 2-adrenergic receptors in CHO-K1 cells (32). Downregulation of the wild-type AT1 receptor remained unperturbed by pertussis toxin treatment (data not shown), confirming that coupling to Gi is not necessary for AT1 receptor downregulation. Of note, the early phase of downregulation of the Gq-uncoupled receptors AT1-i2 LOOP and AT1-D74N was also unaffected by pertussis pretreatment (data not shown), confirming no apparent role for Gi in AT1 receptor downregulation.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The present study focused on the ability of AT1 receptors to undergo downregulation during the initial 24 h of ANG II exposure. Previous studies focused primarily on the role of decreased receptor production in AT1 receptor downregulation but concluded that an internalization-degradation pathway also contributes to downregulation (1, 24). To date, no studies have examined the latter process. It is commonly assumed that this process involves or requires endocytosis of the AT1 receptor (24). Our data show that endocytosis-deficient mutant receptors (AT1-309T and AT1-311T) remained fully capable of rapid downregulation comparable to the wild-type AT1 receptor. This finding suggests that minimal, if any, endocytosis is necessary for downregulation. Furthermore, the AT1-i2 LOOP and AT1-D74N mutants displayed endocytosis comparable to wild-type AT1 receptor but exhibited an impaired ability to undergo downregulation. Taken together, these data indicate that endocytosis is neither necessary nor sufficient for AT1 receptor downregulation.

The finding that endocytosis-deficient mutants are capable of efficient downregulation suggests either the existence of an alternative mechanism for internalizing AT1 receptors for degradation or degradation of receptors on the cell surface. The existence of an alternative mechanism for internalizing AT1 receptors is consistent with the observation of Zhang et al. (33) that the AT1 receptor is capable of clathrin-dependent and -independent internalization when cotransfected with mutant dynamin in HEK-293 cells. Dissociation of receptor endocytosis and downregulation have also been observed in the beta 2-adrenergic receptor, but no alternative pathway for downregulation has been proposed (4, 10, 31). Recent studies have identified caveolae as an additional means of internalizing ligand-receptor complexes (8, 21, 22, 26). The CCK and B2 bradykinin receptors appear to utilize the following dual pathways for internalization: endocytosis and caveolae (8, 21, 22, 26). The behavior of the AT1-309T and AT1-311T receptors may be consistent with the presence of a caveolar pathway for AT1 receptor internalization and degradation. The recent observation that caveolin-1 coimmunoprecipitates with the AT1 receptor after brief agonist stimulation of VSMCs (14) may support this hypothesis, but the present study did not address this possibility.

Degradation of receptors on the cell surface is an alternative explanation for the finding that endocytosis-deficient mutants are capable of downregulation. This hypothesis is consistent with the recent observation that ligand-induced activation of G protein-coupled receptors, such as the endothelin-1 receptor, induces metalloproteinase activity (25). The present study cannot address this possibility, which warrants further study.

Although an alternative pathway for ANG II-induced loss of cell surface receptors appears to be operative for the truncated receptors, the present study does not exclude the possibility that a classical endocytotic pathway is the primary means of removing surface receptors when endocytotic capability is preserved, as in the wild-type AT1 receptor. The AT1-D74N mutant may prove particularly useful in answering this question. This mutant displayed a biphasic pattern of downregulation. During the initial phase of downregulation (0-4 h), loss of AT1-D74N receptors paralleled that of wild-type AT1 receptor. Because endocytosis was preserved in this mutant, it is possible that this initial phase of receptor loss represents either clathrin-dependent or clathrin-independent internalization of surface receptors, leading to downregulation. During the subsequent phase of downregulation (4-24 h), the AT1-D74N mutant recovered binding sites and returned to baseline levels of receptor density. The mechanism by which AT1-D74N restored binding sites remains unknown. Future investigations of this construct may prove insightful in further defining the mechanisms of AT1 receptor downregulation.

The findings of the present study do not address the role of decreased receptor production in AT1 receptor downregulation. Previous studies in both VSMC and adrenal fasciculata cells described time- and dose-dependent reductions in AT1 receptor mRNA induced by ANG II (1, 17, 24). These studies noted a rapid time course of AT1 receptor downregulation that corroborated our findings (1, 17, 24). By comparison, Ouali et al. (24) noted that prevention of new receptor production with the translational inhibitor cycloheximide resulted in a gradual loss of AT1 receptors in adrenal fasciculata cells, yielding a receptor half-life of 14.8 h. Thus decreasing receptor production impacts AT1 receptor density, but much less rapidly than the 3-h half-life induced by ANG II treatment (24). This time course discrepancy suggests that rapid internalization and degradation of receptors plays an integral part in AT1 receptor downregulation. The observations of Ouali et al. (24) and those of the present study do not, however, exclude a role for reduced receptor production in AT1 receptor downregulation. Using a recombinant AT1 receptor expressed in A7r5 VSMCs, Adams et al. (1) concluded that regulation of AT1 mRNA levels in VSMC plays a crucial role in dictating the degree to which AT1 receptor downregulation will occur. The degree to which each of these processes contributes to AT1 receptor downregulation remains unresolved primarily because of the difficulty in separating the two processes experimentally (1, 24).

An additional goal of our study was to determine whether G protein coupling is required for AT1 receptor downregulation. Our experiments demonstrated for the first time that G protein coupling may be necessary for the terminal phase of AT1 receptor downregulation. The AT1-i2 LOOP and AT1-D74N mutants displayed a biphasic pattern of downregulation. The initial phase of downregulation (2-4 h) was entirely independent of Gq coupling, since the AT1-i2 LOOP mutant proved to be indistinguishable from wild-type AT1 despite complete uncoupling from Gq. The second phase of downregulation, however, appears to require coupling to Gq since both mutants with impaired coupling to Gq (AT1-i2 LOOP and AT1-D74N) were unable to undergo further downregulation after the initial 2-4 h of downregulation. In addition, pretreatment with pertussis toxin had no effect on wild-type AT1 downregulation, so coupling to Gi is not required for AT1 receptor downregulation. Taken together, these data suggest that coupling to Gq, but not Gi, may be necessary for the apparent second phase of AT1 receptor downregulation. Prior attempts to determine if signaling is necessary for AT1 downregulation have been inconclusive. Lassegue et al. (17) concluded that protein kinase C activation was not necessary for AT1 downregulation, whereas Ouali et al. (24) found that protein kinase C activation downregulated AT1 mRNA but not binding sites. The present study provides the most direct evidence to date that AT1 receptor downregulation is at least partially dependent on G protein (Gq) coupling.

In summary, our data indicate that AT1 receptor downregulation is much more complex than previously hypothesized. In addition to the proposed role of decreased receptor production in downregulation, there appears to be a mechanism for degrading existing AT1 receptors. Although this mechanism may involve an endocytotic pathway, data from the endocytosis-deficient mutants suggest the existence of an alternative pathway for clearance and degradation of cell surface receptors that does not require classical clathrin-dependent endocytosis. The identity of this pathway remains unknown. The current studies further suggest that G protein coupling is necessary for the terminal phase of AT1 receptor downregulation. The present study used a transfected cell system, so it remains to be determined whether these results may be extrapolated to native cell lines, such as VSMCs. Continued pursuit of a mechanistic understanding of AT1 downregulation, using both transfected cell systems and native cell lines, is critical to understanding cellular homeostasis, adaptive responses, and pathological expression of the AT1 receptor.


    ACKNOWLEDGEMENTS

This work was supported by Department of Veterans Affairs New Investigator Award VISN 17 (to J. G. Modrall) and National Heart, Lung, and Blood Institute Grant HL-46417 (to R. R. Neubig).


    FOOTNOTES

Address for reprint requests and other correspondence: J. Gregory Modrall, Dept. of Surgery, Univ. of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75390-9157 (E-mail: greg.modrall{at}utsouthwestern.edu).

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 1 September 1999; accepted in final form 4 April 2001.


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