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Am J Physiol Endocrinol Metab 290: E840-E848, 2006. First published December 6, 2005; doi:10.1152/ajpendo.00458.2005
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The AT1A receptor "gain-of-function" mutant N111S/{Delta}329 is both constitutively active and hyperreactive to angiotensin II

Sandrine Billet, Sabine Bardin, Rachida Tacine, Eric Clauser, and Sophie Conchon

Département d'Endocrinologie, Institut Cochin; Institut National de la Santé et de la Recherche Médicale, U567; Centre National de la Recherche Scientifique Unité Mixte de Recherche 8104; and Faculté de Médecine René Descartes, Université Paris Descartes, Unité Mixte de Recherche et de Service 8104, Paris, France

Submitted 21 September 2005 ; accepted in final form 30 November 2005


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 GRANTS
 REFERENCES
 
The renin-angiotensin-aldosterone system (RAAS) is central to cardiovascular and renal physiology. However, there is no animal model in which the activation of the RAAS only reflects the activation of the angiotensin II (ANG II) AT1 receptor. As a first step to developing such a model, we characterized a gain-of-function mutant of the mouse AT1A receptor. This mutant carries two mutations: N111S predicted to activate the receptor constitutively and a COOH-terminal deletion, {Delta}329, expected to reduce receptor internalization and desensitization. We expressed this double mutant (AT1A-N111S/{Delta}329) in heterologous cells. It showed a pharmacological profile consistent with that of other constitutively active mutants. Furthermore, it increased basal production of inositol phosphates, as well as basal cytosolic and nuclear ERK activities. Basal proliferation of cells expressing the mutant was also greater than that of the wild type. The double mutant was poorly internalized and failed to recruit beta-arrestin 2 in the presence of ANG II. It also showed hypersensitive and hyperreactive responses to ANG II for both inositol phosphate production and ERK activation. The additivity of the phenotypes of the two mutations makes this mutant an appropriate candidate to test the physiological consequences of the AT1A receptor activation itself in transgenic animal models.

G protein-coupled receptor; hyperreactivity; beta-arrestin; extracellular signal-regulated kinase

THE AT1 RECEPTOR IS A G PROTEIN-COUPLED RECEPTOR (GPCR) and the principal cellular effector of the renin-angiotensin-aldosterone system (RAAS). Activation of this receptor by the octapeptide angiotensin II (ANG II) results in contraction of vascular smooth muscle cells, aldosterone secretion by the adrenal cortex, and regulation of the water-electrolyte balance by the kidney. The classical signaling pathway of the AT1 receptor involves initial coupling to a heterotrimeric Gq/11 protein and activation of phospholipase Cbeta, which generates inositol 1,4,5-trisphosphate (IP3) and diacylglycerol. IP3 mobilizes intracellular calcium stores, and diacylglycerol activates protein kinase C (PKC). After binding ANG II, the AT1 receptor is phosphorylated, associates with beta-arrestins, and is internalized into vesicles, which traffic through the endosomal pathway before recycling (26). Hypertrophic and hyperplasic effects of ANG II have also been described in several target tissues (4, 9, 23). These effects are mediated by the AT1 receptor and are believed to involve the mitogen-activated protein (MAP) kinase signaling pathway, which is activated either by direct action of PKC or through the scaffolding role of the beta-arrestin-2 (28) or by transactivation of the EGF receptor (23), depending on the cell type. Pharmacological and mutation studies of the AT1 receptor have largely deciphered the molecular mechanisms of its activation and regulation, identifying several types of both loss- and gain-of-function mutations. However, none of these mutations has been found in human pathology (8), unlike what is observed for several other GPCRs (opsins, vasopressin, LH, FSH, or parathyroid hormone receptors) (5, 17, 20, 24). Because of the crucial role of ANG II in cardiovascular function, it would be valuable to select a gain-of-function AT1 mutant and analyze its phenotype in transgenic animals. Two types of gain-of-function mutations could be selected or associated: constitutive activity and absence of desensitization of the receptor. Several point mutations resulting in constitutive activation of the receptor have been described (11, 18). One of the most potent mutations is the substitution of the asparagine 111 in the third transmembrane domain by a serine (N111S). This mutant activates Gq protein in basal conditions, as demonstrated by the increased production of the second-messenger IP3 in the absence of ANG II (11). ANG II stimulates the IP3 production via this mutant to the same extent as via the wild-type (wt) receptor, but downstream desensitization of the signaling pathway has recently been reported (2). Interestingly, this mutant is also constitutively internalized and accumulates in intracellular vesicles (3, 16). These observations suggest that constitutively active mutants of the AT1 receptor are also partly constitutively desensitized. We therefore combined this mutant with a second gain-of-function mutation, to reduce the desensitization of the receptor. Deleting part (30 amino acids) of the COOH-terminus of the AT1 receptor ({Delta}329 mutant) reduces receptor internalization and desensitization and amplifies intracellular signaling induced by ANG II (7).

The two gain-of-function mutations (N111S and {Delta}329) were introduced into mouse AT1A cDNA, and the resulting double mutant was expressed in heterologous cells. We report here the pharmacology, the trafficking, and the signaling properties of this mutant. Its in vitro phenotype associates both constitutive activity and an amplified intracellular response to ANG II. It is, therefore, a good candidate for animal model investigations of the physiological consequences of a hyperactive AT1 receptor.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 GRANTS
 REFERENCES
 
Constructions. All mouse AT1A receptor constructs were derived from a 7-kb SpeI-SacI genomic fragment of a commercial BAC clone (no. FBAC4432, Incyte Genomics, St. Louis, MO).

The coding sequence of AT1A-wt was amplified by PCR from the genomic fragment with two primers (primer 1: 5'-GATCAAGCTTGTGTCTGAGACCAACTCAACCC-3'; primer 2: 5'-GATCGGATCCTCACTCCACCTCAGAACAAGAC-3') and inserted into a modified version of the Clontech pTRE vector (18) using the HindIII and BamHI enzymes.

The double-mutant AT1A-N111S/{Delta}329 was obtained by site-directed mutagenesis using sequential PCR. First, two mutated fragments of the AT1A coding sequence were synthesized by two parallel PCR reactions. The 5' fragment (PCR A) was amplified with a sense primer starting in the 5' genomic region (primer 3: 5'-CCACAACAGTACAACAATCTCC-3') and an antisense primer containing the N111S mutation (primer 4: 5'-GCTGGCGTAGAGGCTGAAACTGACGCT-3'). The 3' fragment (PCR B) was amplified with a sense primer complementary and antiparallel to primer 4 (primer 5: 5'-AGCGTCAGTTTCAGCCTCTACGCCAGC-3') and an antisense primer, which introduced a stop codon at position 329 (primer 6: 5'-ATCGAGCTCTTATGAGTGCGACTTGGCCTTTGGG-3'). In a second step, PCR A and B were mixed and amplified with primers 3 and 6 to obtain the entire coding sequence.

Finally, the entire coding sequence obtained with primers 3 and 6 was amplified with primer 1 and primer 7 (5'- GATCGGATCCGAGCTCTTATGAGTGCGACTTG-3') to introduce restriction sites appropriate for insertion into the pTRE vector. The wt and mutated sequences were verified by DNA sequencing.

Cell culture and transfection. Human embryonic kidney (HEK)-293 (ATCC F-14742,1573-CRL) and Chinese hamster ovary (CHO) Tet-Off (Clontech) cells were grown at 37°C in 5% CO2 in MEM and Ham's F-12 medium, respectively, supplemented with 7.5% fetal calf serum (FCS), 0.5 mM glutamine, and 100 U/ml penicillin. Geneticin (100 µg/ml G418, Invitrogen) was added to CHO medium for Tet-Off selection.

Transient transfections were performed using lipofection reagents: Dosper (Boehringer Mannheim) for HEK-293 and Fugene 6 (Roche) for CHO cells, both according to the manufacturer's recommendations. Transfected cells were used 36–60 h after transfection.

Cell lines CHO-TetOff-AT1A-wt and CHO-TetOff-AT1A-N111S/{Delta}329 stably expressing AT1A receptors were established by cotransfecting pTRE-AT1A-wt or pTRE-AT1A-N111S/{Delta}329 with a hygromycin selection marker (29) using the Effecten (Qiagen) transfection reagent, according to the manufacturer's recommendations. Expression of the AT1A-wt and the mutant AT1A-N111S/{Delta}329 was confirmed by receptor binding assays. The pure clonal cell lines were then grown under G418 (100 µg/ml) and hygromycin (200 µg/ml) selections.

For all experiments on these stable cell lines, doxycycline (2 µg/ml) was included in media to repress the expression of the receptors; it was removed at least 7 days before each experiment to allow membrane expression of the receptors.

All experiments were performed on cells expressing comparable numbers of either AT1A-wt or AT1A-N111S/{Delta}329 receptors.

Binding assays. Saturation and competition binding assays with 125I-labeled ANG II were performed on intact cells, as described previously (6). Competition binding assays were performed using 4 nM 125I-ANG II and a series of concentrations (10–11 to 10–4 M) of the various ligands. Each experiment was performed in triplicate. Binding data were analyzed by linear regression with the Prism program (GraphPad Software).

Internalization assays. Internalization of the wt and mutant AT1A was measured as the percentage of 125I-ANG II resistant to acid wash, as described previously (6). Transfected cells were incubated with 0.4 nM 125I-ANG II in binding buffer with or without 1 µM ANG II for 3 h at 4°C, washed twice, and incubated in binding buffer alone at 37°C for various time periods. The samples were then cooled to 4°C and acid washed (0.2 M acetic acid, 0.5 M NaCl in binding buffer, 5 min at 4°C), and the cells were lysed with 1 M NaOH. Surface-bound and internalized fractions of 125I-ANG II were then determined.

Confocal microscopy. CHO cells stably expressing the wt or the mutant AT1A receptors were transiently transfected with a beta-arrestin-2-enhanced green fluorescent protein (eGFP) construct (21) (500 ng per petri dish) using Fugene 6. The day after transfection, the cells were seeded into Labtek 8-well chambered coverglass (Nunc). Two days later, the cells were examined under a Leica TCS SP2 AOBS laser scanning confocal microscope at 37°C. Images were obtained with a x63 oil-immersion lens and recorded every minute for the first 5 min and then every 5 min after the addition of 100 nM ANG II to the samples.

Inositol phosphate production. ANG II-stimulated inositol phosphate (IP) production was determined as described previously (6). Cells were cultured in 24-well plates (50–70% confluence), labeled with 2 µCi/ml [myo-3H]inositol for 24 h in inositol-free MEM, and incubated with various concentrations of ANG II for 30 min in the presence of 10 mM LiCl. The IP content was determined by methanol extraction and separation on a Dowex anion exchange resin (AG1-X8 Bio-Rad).

ANG II binding was measured in parallel, to verify the equivalent number of binding sites in the two transfected cell lines.

ERK-1/2 phosphorylation. ERK phosphorylation was measured in transfected CHO cells in the presence or absence of ANG II or ANG IV and of the PKC inhibitor Ro31-8425 (5 µM) added 20 min earlier. Cells were grown in 6-well or 100-mm plates for 16 h in a starvation medium (F-12 medium with L-glutamine, penicillin, streptomycin, 0.1% BSA, 25 mM HEPES, pH 7.5) and then stimulated at 37°C either for various times with a given concentration of agonist or for 5 min with various concentrations of agonist. The samples were rinsed once in cold PBS, and Laemmli buffer was directly added. The lysates were collected, boiled, and centrifuged.

In some cases, cells were scraped off in a lysis buffer [10 mM Tris, pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.3% IGEPAL, 1 mM PMSF, protease inhibitor complete (Roche)] and centrifuged for 5 min at 500 g. The supernatant (cytosolic fraction) was collected, and the pellets were washed once with lysis buffer without IGEPAL and resuspended in lysis buffer (nuclear fraction). Aliquots of 50 µg of protein of these fractions in Laemmli buffer were boiled, centrifuged, and tested for purity by immunoblotting with anti-{alpha}-actin (1:1,000, Santa Cruz) and anti-Lamins A/C (1:1,000, Santa Cruz) antibodies.

Cellular protein extracts were separated on 10% acrylamide SDS-PAGE gels and subjected to Western blotting, according to standard procedures using either anti-phospho-ERK1/2 antibody (1:1,000, Cell Signaling Technology) or anti-ERK1/2 antibody (1:10,000, Upstate), and an enhanced chemiluminescence kit (supersignal Westpico, Pierce). ChemiGenius (Syngen), with the GeneSnap and GeneTool software, was used to acquire the signals and measure ERK phosphorylation.

Elk1 luciferase reporter assay. ERK1/2-dependent transcription was measured using an Elk1-driven luciferase reporter system (22). CHO cells expressing the receptors were transiently transfected with the plasmids GAL4-Elk1 (5 ng/100 mm2 plate), pFR-Luc (1 µg/100 mm2 plate), and pCH110-betaGal (500 ng/100 mm2 plate). The total amount of DNA added to each sample was adjusted to 2 µg per dish with an empty vector. The GAL4-Elk1 plasmid encodes a fusion protein containing the GAL4 DNA binding domain and the transactivation domain of Elk1. pFR-Luc encodes the firefly luciferase gene under the control of the GAL4 DNA-binding element. One day after transfection, cells were transferred to 6-well dishes, and 1 day later they were starved of serum for 16 h. They were then stimulated with 100 nM ANG II for 6 h. The cells were lysed, and firefly luciferase and beta-galactosidase activities in the cell lysates were assayed. Firefly luciferase activities were normalized to beta-galactosidase activities.

Bromodeoxyuridine incorporation. Cells were seeded on coverslips placed in FB12 plates (1,000 cells per 13-mm2 coverslip) in complete medium. One day later, cells were washed with PBS and starved of serum. After 2 days, ANG II was added to a final concentration of 100 nM. This ANG II stimulation was repeated every day for 3 days. On the last day, the cells were incubated for 4 h with bromodeoxyuridine (BrdU; 6 µg/ml, Roche), washed, and fixed in 4% paraformaldehyde at room temperature for 20 min. The samples were then boiled for 10 min in citrate buffer and immunostained with monoclonal anti-BrdU antibody (Roche) and Alexa Fluor 488 anti-mouse antibody (Molecular Probes). Cells were stained with 4,6-diamidino-2-phenylindole (Fluka) and mounted. The percentage of BrdU incorporation was determined by counting the number of BrdU-positive nuclei among at least 600 nuclei in different fields.

Data analysis. Data points are means ± SE of at least three independent experiments. ANOVA followed by the Fisher least significant difference a posteriori test was carried out to study differences between groups using the StatView program. Curves were fitted with the Prism program (GraphPad Sofware).


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
GRANTS
 REFERENCES
 
In this study, a gain-of-function mutant of the ANG II AT1 receptor was characterized in cellular models with the final goal to analyze its physiological phenotype in animal models. This mutant associates a constitutively activating mutation (N111S) with a deletion of the COOH terminus of the receptor ({Delta}329). Constitutively active mutants are classically downregulated, due to constitutive internalization (16) and desensitization of the signaling pathways (2). Therefore, association of the COOH-terminal deletion, which reduces internalization and desensitization, may strengthen the phenotype. In vitro characterization of this double mutant demonstrates that the two phenotypes are additive.

Pharmacological properties of the AT1A double mutant N111S/{Delta}329. The pharmacological properties of the AT1A-N111S/{Delta}329 double mutant were compared with those of the wt receptor after transient transfection of HEK-293 cells and stable transfection of CHO cells (Table 1). In saturation binding studies with the radioactive agonist 125I-ANG II, the wt and mutant receptors had comparable affinity for the hormone (Kd = 2.45 ± 0.58 vs. 2.14 ± 0.18 nM). The Kd of the double mutant was similar to those reported for the single mutants AT1A-{Delta}329 (7) and AT1A-N111S (11).


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  		Table 1. Pharmacological properties of the wild-type AT1A-wt and the AT1A-N111S/{Delta}329 mutant in HEK-293 cells and stably transfected CHO TetOff cells

 
We then studied the binding characteristics of several compounds in 125I-ANG II competition binding experiments. The wt and mutant receptors had similar Ki values for the peptide agonist [Sar1]ANG II and antagonist [Sar1,Ile8]ANG II in both HEK-293 and CHO cells. However, the Ki value for a nonpeptide biphenyltetrazol inverse agonist Losartan was about 40 and 70 times higher for the AT1A-N111S/{Delta}329 mutant than for the wt receptor in HEK and CHO cells, respectively. In contrast, the peptide partial agonist ANG IV [angiotensin (3–8)] had a very low affinity for the wt AT1 receptor (7853.03 nM), but a higher affinity for AT1A-N111S/{Delta}329 (34.70 nM). Similarly, the AT2-specific pseudopeptide ligand CGP42112A had a fivefold higher affinity for the mutant than wt receptor in both cell types. This modified pharmacological profile of the AT1A-N111S/{Delta}329 mutant is consistent with previously described profiles of constitutively active AT1 mutants (18). Nonpeptide inverse agonists of the biphenyltetrazol class (including Losartan) have less affinity for constitutively activated AT1A mutants, particularly those affecting residue N111. This is probably due to a conformational change affecting the binding site of the biphenyltetrazol moiety. In addition, peptide (ANG IV) or pseudopeptide (CGP42112) partial agonists of the AT1A receptor have greater affinity for mutants, which mimic an active conformational state and are indeed full agonists in this situation (14). Finally, the {Delta}329 mutation by itself or in the context of this double mutant does not modify the binding properties of the receptor (7).

AT1A-N111S/{Delta}329 internalization and beta-arrestin 2 translocation. The AT1A-N111S/{Delta}329 mutant was poorly internalized following ANG II binding (maximal internalization 23.88 ± 3.24% compared with the wt value of 74.55 ± 3.71%, Fig. 1A), as described for the {Delta}329 mutant (7). The interaction of GPCR with beta-arrestins is a major event for both receptor internalization and receptor signaling (15). We studied by confocal microscopy the intracellular localization of an eGFP-tagged beta-arrestin-2 in living CHO cells expressing the wt or mutated receptors, after ANG II stimulation (Fig. 1B). From a basal cytosolic localization, beta-arrestin-2-eGFP was recruited to the plasma membrane 5 min after addition of ANG II, presumably by interacting with the AT1A receptor. This interaction, well documented by others (10, 12), is very stable and persists during the endocytic routing of the AT1 receptor, with some intracellular vesicles visible 10 min and a perinuclear localization 20 min after ANG II stimulation. In cells expressing the AT1A-N111S/{Delta}329 mutant, the beta-arrestin-2-eGFP was cytosolic in basal conditions and remained mainly cytosolic after stimulation by ANG II. Moreover, no vesicles containing beta-arrestin-2-eGFP were observed. Similar results have been reported for a slightly shorter truncated mutant ({Delta}324) (13, 25, 26). This {Delta}324 truncated mutant has only a low-affinity interaction with beta-arrestin-2, which weakly and transiently translocates to the plasma membrane without persisting association in the endocytic vesicles (27). In conclusion, this result indicates that constitutive internalization of the N111S mutation is completely occulted by the internalization defect resulting from the COOH-terminal deletion.


Figure 1
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  		Fig. 1. Cellular trafficking of the wild-type (wt) and mutant AT1A receptor and of beta-arrestin-2 after ANG II stimulation. A: time course of ANG II-induced receptor internalization. Cells expressing AT1A-wt ({square}) or AT1A-N111S/{Delta}329 (bullet) were prelabeled with 125I-ANG II at 4°C for 3 h. Cells were then washed and incubated at 37°C for various times to allow internalization. Noninternalized tracer was removed by acid washing, and internalized tracer levels were determined after NaOH treatment. Results are expressed as percentage of total specific binding, and means ± SE of 3 independent experiments performed in duplicate are shown. *Significantly different from wt values, P < 0.05. Imax, maximal internalization. B: translocation of beta-arrestin-2-enhanced green fluorescent protein (eGFP) induced by ANG II activation of wt or mutant AT1A receptors. Cells expressing AT1A-wt or AT1A-N111S{Delta}329 were transiently transfected with an expression vector encoding beta-arrestin-2-eGFP. The recruitment of beta-arrestin-2-eGFP was examined by confocal microscopy for 20 min following stimulation with 100 nM ANG II.

 
AT1A-N111S/{Delta}329 intracellular signaling. Following ANG II binding, the AT1A receptor interacts with various intracellular partners to elicit cellular responses to the hormone. IP production is a marker of the main signaling pathway of the receptor, which involves the heterotrimeric Gq protein, the phospholipase Cbeta, and leads to calcium signaling and PKC activation. ANG II-induced IP production was characterized in both HEK (Fig. 2) and CHO cells expressing similar levels of wt or AT1A-N111S/{Delta}329 mutant. The basal IP production was significantly increased in cells expressing the mutant receptor compared with the wt receptor (44.35 vs. 20.88% of maximal IP production in wt cells for HEK and 29.33 vs. 19.94% for CHO cells). The ANG II-induced IP production through the mutant receptor was significantly higher (150.33% of maximum IP production in wt cells for HEK and 161% for CHO cells), and the EC50 was significantly lower (one log in both cell types). Therefore, the AT1A-N111S/{Delta}329 mutant activates this signaling pathway both constitutively and with increased sensitivity to ANG II.


Figure 2
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  		Fig. 2. Dose-dependent ANG II stimulation of inositol phosphate (IP) production in human embryonic kidney (HEK)-293 cells expressing the wt ({square}) or the mutant (bullet) receptors. Results are means ± SE of 3 independent experiments and are expressed as percentages of the maximal stimulation for the wt receptor. Significantly different from wt values: *P < 0.05, **P < 0.01.

 
ERK1/2 phosphorylation via the AT1A-N111S/{Delta}329 receptor. To describe the cell growth properties associated with the gain-of-function double mutant, we compared MAP kinase activation in cells expressing either wt or mutant AT1 receptors. ANG II induces ERK1/2 phosphorylation via the AT1 receptor using G protein-dependent and -independent pathways, depending on the cell type. We were unable to obtain reproducible results in transiently transfected HEK-293 cells. Consequently, stable cell lines were established. Surprisingly and despite several attempts, we were unable to establish clones expressing the AT1A-N111S/{Delta}329 mutant using standard viral-derived expression systems until we used the tetracycline-inducible expression system TetOff (Clontech). CHO cells transfected with the wt or the mutant AT1 receptor were grown in the presence of doxycyline, which turns off the transcription of the receptor genes. One week before the experiments, doxycycline was removed to allow receptor expression.

ANG II stimulated ERK phosphorylation dose dependently, in CHO expressing either the AT1-wt or the AT1-N111S/{Delta}329 receptors (Fig. 3A). However, the maximal response with the AT1A-N111S/{Delta}329 receptor was almost double (190 ± 17%) of the effect observed for the wt receptor, and the EC50 of this effect was significantly lower than that for the wt receptor (0.29 ± 0.06 vs. 3.98 ± 1.48 nM).


Figure 3
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  		Fig. 3. Stimulation of ERK1/2 phosphorylation by ANG II (A) and ANG IV (B). Chinese hamster ovary (CHO) cells expressing AT1A-wt ({square}) or AT1A-N111S/{Delta}329 (bullet) were starved of serum for 16 h and stimulated with ANG II or ANG IV for 5 min at 37°C. The top panels show representative immunoblots of phospho-ERK1/2 (p-ERK1/2) and total ERK1/2 (ERK1/2). ERK1/2 phosphorylation was quantified by densitometry, normalized to the amount of total ERK1/2 in each lane, and expressed as a percentage of the maximal phosphorylation of ERK1/2 in cells expressing the wt AT1A receptor. Results are means ± SE of 3–4 independent experiments. *Significantly different from wt values, P < 0.05.

 
ANG IV (Fig. 3B) was a very weak and partial agonist for inducing ERK phosphorylation via the AT1A-wt receptor (EC50 = 1,054 ± 481 nM) but was a fully active agonist via the AT1A-N111S/{Delta}329 receptor (EC50 = 3.76 ± 3.08 nM). Thus the N111S/{Delta}329 mutant mediates an ERK activation signal in response to ANG II, which is both amplified ({Delta}329) and pharmacologically specific to constitutively active mutants (N111S).

In conclusion, the maximal response of cells expressing the AT1A-N111S/{Delta}329 receptor to ANG II was 1.5 to 2 times higher than that of the wt and with a greater sensitivity (10 times lower EC50) for both IP production and ERK phosphorylation. In addition, the mutant receptor has a major internalization defect. Therefore, it is likely that the desensitization of this mutant is also defective, leading to its hyperreactive phenotype.

Time course of ANG II-induced ERK1/2 phosphorylation. We measured ERK phosphorylation at various times after stimulation with 100 nM ANG II (Fig. 4). Despite confirmation of the stronger response mediated by the mutant receptor, the time course of ERK phosphorylation in response to ANG II was the same for mutant and wt receptors: a transient peak after 5 min followed by a plateau that lasted for at least 60 min. Therefore, the impaired desensitization of the mutant receptor leads to amplified activation of ERK1/2 but does not modify the kinetic pattern of this activation. In both cell lines, the initial peak and the following plateau were reduced by >80% by a PKC inhibitor (Ro31-8425) (Fig. 4A).


Figure 4
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  		Fig. 4. Time course of ERK1/2 stimulation by ANG II with or without the PKC inhibitor Ro31-8425. CHO cells expressing AT1A or AT1A-N111S/{Delta}329 were serum starved for 16 h and then treated with DMSO only or 5 µM Ro31-8425. After 20 min, cells were stimulated with 100 nM ANG II for various time periods at 37°C, and cellular extracts were prepared and immunoblotted. A, top: representative immunoblots. ERK1/2 phosphorylation was quantified by densitometry, normalized to the amount of total ERK1/2 in each lane, and expressed as a percentage of the maximal stimulation for each cell type without Ro31-8425 (A) or as a percentage of the maximal stimulation for the wt receptor without Ro31-8425 (B). Results are the means ± SE of 5 experiments. *Significantly different from wt values, P < 0.05.

 
The classical kinetic of ANG II-activated ERK1/2 phosphorylation is a biphasic curve, the initial peak (5 min) of which is PKC dependent, and the subsequent plateau (up to 90 min) is beta-arrestin dependent in HEK-293 cells (1). Therefore, the present data suggest that the beta-arrestin pathway of ERK activation is not involved in CHO cells: 1) the secondary plateau is much lower than the initial peak; 2) both are almost completely inhibited by the PKC inhibitor Ro31-8425; 3) overexpression of beta-arrestin 2 did not modify the time course of ERK phosphorylation in these cells (data not shown);and 4) the AT1A-N111S/{Delta}329 mutant, which does not interact with beta-arrestin, has a similar pattern of ERK phosphorylation. Therefore, the CHO cell line may not be a suitable model to study the beta-arrestin-2-dependent ERK activation of the receptor.

The AT1A-N111S/{Delta}329 receptor constitutively activates ERK1/2. In the experiments described above, the basal phosphorylation of ERK1/2 was slightly but not significantly higher in the cells expressing the mutant receptor than in those expressing similar amounts of wt receptor. Basal ERK phosphorylation was measured in cytosolic and nuclear fractions of transfected cells to study the potential constitutive activitation of the ERK pathway (Fig. 5A). In these basal conditions, the cytosolic fraction of the mutant cells contained significantly more phosphorylated ERK1/2. However, no phosphorylated ERK was detected in the nuclear fractions of either the wt or the mutant cells.


Figure 5
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  		Fig. 5. Basal ERK1/2 phosphorylation in CHO cells expressing the AT1A or AT1A-N111S/{Delta}329 receptor. Cells were serum starved for 16 h and stimulated with 100 nM ANG II for 5 min at 37°C. A: nuclear and cytosolic fractions were prepared, and their purity was assessed by immunoblotting with anti-{alpha}-actin or anti-Lamins A/C (Santa Cruz) antibodies. The amount of ERK1/2 was determined by immunobloting with a p-ERK1/2-specific antibody. B: ERK1/2-dependent transcription was measured using the Elk1-driven luciferase reporter system (see MATERIALS AND METHODS). Results are means ± SE for 3 experiments, and values are expressed as multiples of the wt basal value. *Significantly different from wt, P < 0.05.

 
We used an Elk1 reporter system to analyze the transcriptional activity of phospho-ERK1/2 (Fig. 5B, see MATERIALS AND METHODS). In basal conditions, Elk1-GAL4 activation was significantly higher in CHO AT1A-N111S/{Delta}329 than CHO AT1A cells (3.11 ± 0.71 times higher), despite the presence of similar numbers of receptors. After stimulation by 100 nM ANG II, the transcriptional response of the mutant receptor was approximately double that of the wt receptor. These results demonstrate that the N111S/{Delta}329 mutant constitutively activates the MAP kinases ERK1/2, both in the cytosol and in the nucleus.

These data confirm constitutive activity of the mutant on another downstream signaling pathway activated by ANG II, the MAP kinase cascade. Most techniques for detecting the activation of ERK1/2 on whole cell extracts are not sensitive enough to detect differences in serum-deprived conditions. Therefore, we used subcellular fractionation and an ERK-dependent transcriptional activity test to demonstrate a significant increase of phospho-ERK1/2 in both the cytosol and the nucleus of CHO cells expressing the AT1A-N111S{Delta}329 receptor. The apparent discrepancy between the immunoblot findings for nuclear fractions and the transcriptional assay is probably due to the different sensitivities of the two techniques. Indeed, the immunoblot gives an almost instant image of the nuclear phospho-ERK1/2 content, whereas the transcriptional activity test measures the accumulation of the transcript over a period of 6 h.

Mitogenic activity of the AT1A-N111S/{Delta}329 receptor. We studied the mitogenic properties of CHO cells expressing similar numbers of AT1A-wt or mutant AT1A-N111S/{Delta}329 receptors by measuring BrdU incorporation into the DNA of dividing cells in various conditions (Fig. 6). In normal culture conditions with FCS, BrdU incorporation was similar in both cell lines (data not shown). In the absence of FCS, the cells expressing the AT1A-N111S/{Delta}329 receptor proliferated significantly faster than wt cells (10.12 ± 1.43% of cells were BrdU positive vs. 5.34 ± 0.54%). However, following stimulation by 100 nM ANG II for 3 days, the proliferative response of both cell lines was comparable (15.33 ± 1.70% for AT1A, 16.77 ± 1.51% for AT1A-N111S/{Delta}329).


Figure 6
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  		Fig. 6. Cell proliferation of CHO cells expressing AT1A or AT1A-N111S/{Delta}329 receptors. Cells were starved of serum and stimulated for 3 days with 100 nM ANG II. Then they were incubated with bromodeoxyuridine (BrdU) and immunostained using anti-BrdU antibody. The percentage of BrdU incorporation was determined by counting the BrdU-positive nuclei among at least 600 nuclei in distinct fields. DAPI, 4,6-diamidino-2-phenylindole. *Significantly different from wt values, P < 0.05.

 
Therefore and despite clear hyperreactivity and hypersensitivity of the mutant to ANG II for both the IP production and MAP kinase phosphorylation, there is no increase of the maximal response to ANG II for cell division. This result suggests that the hyperreactive signaling of the mutant may be attenuated by secondary regulatory mechanisms, previously analyzed in culture of glomerulosa cells (19), at least for this long-term action of ANG II. Whether or not this mutant is able to transduce a hyperreactive signal for all or only part of the physiological actions of ANG II will be better investigated in an animal model expressing this mutant.

In conclusion, the AT1A-N111S/{Delta}329 mutant presents several phenotypic traits of constitutive activity: a specific pharmacological profile, increased basal IP production, ERK phosphorylation, and proliferation. This mutant is also hyperreactive to ANG II, as shown by the amplified IP response and ERK phosphorylation following ANG II stimulation. These two gain-of-function phenotypic traits are additive: the constitutive activation of the receptor can result in a "long-term" effect, such as ligand-independent cell proliferation, whereas hyperreactivity gives more short-term amplified responses. This makes possible the development of an animal model expressing such a receptor to study both the long-term effects of this expression and the physiological responses induced by acute stimulations of the RAAS.


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 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
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This work was supported by Institut National de la Santé et de la Recherche Médicale, Centre National de la Recherche Scientifique, and Université René Descartes, and by grants from Ligue Nationale Contre le Cancer, Association de la Recherche sur le Cancer, Fondation de France, and Societe Française d'Hypertension Arterielle.


   ACKNOWLEDGMENTS
 
We are grateful to M. G. Scott for the beta-arrestin-2-eGFP plasmid, as well as F. Porteu for all of the Elk Luciferase Reporter Assay plasmids. We also thank P. Bourdoncle and F. Letourneur from the confocal and the sequencing facilities of the Institut Cochin.


   FOOTNOTES
 

Address for reprint requests and other correspondence: E. Clauser, Institut Cochin, Département d'Endocrinologie, Faculté de Médecine Cochin Port Royal, 24 rue du Faubourg Saint Jacques, 75014 Paris, France (E-mail: clauser{at}cochin.inserm.fr)

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.


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