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Endocrinology, doi:10.1210/en.2003-0677
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Endocrinology Vol. 144, No. 12 5277-5284
Copyright © 2003 by The Endocrine Society

The Constitutively Active N111G-AT1 Receptor for Angiotensin II Maintains a High Affinity Conformation Despite Being Uncoupled from Its Cognate G Protein Gq/11{alpha}

Mannix Auger-Messier, Martin Clement, Pascal M. Lanctot, Patrice C. Leclerc, Richard Leduc, Emanuel Escher and Gaetan Guillemette

Department of Pharmacology, Faculty of Medicine, Université de Sherbrooke, Sherbrooke, Quebec, Canada J1H 5N4

Address all correspondence and requests for reprints to: Gaetan Guillemette, Ph.D., Department of Pharmacology, Faculty of Medicine, Université de Sherbrooke, 3001, 12th Avenue North, Sherbrooke, Quebec, Canada J1H 5N4. E-mail: Gaetan.Guillemette{at}USherbrooke.ca.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Asn111, localized in the third transmembrane domain of the AT1 receptor for angiotensin II, plays a critical role in stabilizing the inactive conformation of the receptor. We evaluated the functional and G protein-coupling properties of mutant AT1 receptors in which Asn111 was substituted with smaller (Ala or Gly) or larger residues (Gln or Trp). All four mutants were expressed at high levels in COS-7 cells and, except for N111W-AT1, recognized 125I-Ang II with high affinities comparable to that of the wild-type AT1 receptor. In phospholipase C assays, the four mutants encompassed the entire spectrum of functional states, ranging from constitutive activity (without agonist) for N111A-AT1 and N111G-AT1 to a significant loss of activity (upon maximal stimulation) for N111Q-AT1 and a major loss of activity for N111W-AT1. In Ca2+ mobilization studies, N111W-AT1 produced a weak Ca2+ transient and, unexpectedly, N111G-AT1 also produced a Ca2+ transient that was much weaker than that of the wild-type AT1. The agonist binding affinity of N111W-AT1 was not modified in the presence of GTP{gamma} S, suggesting that this receptor is not basally coupled to a G protein. GTP{gamma} S did not modify the high agonist-binding affinity of N111G-AT1 but abolished the coimmunoprecipitation of Gq/11{alpha} with this constitutively active mutant receptor. These results are a direct demonstration that the N111G-AT1 receptor maintains a high affinity conformation despite being uncoupled from the G protein Gq/11.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE OCTAPEPTIDE HORMONE angiotensin II (Ang II) is the active component of the renin-angiotensin system and exerts a wide variety of physiological effects, including vascular contraction, aldosterone secretion, sodium, and water retention, neuronal activation, and cardiovascular cell growth and proliferation (for reviews see Refs. 1 and 2). Virtually all of the known physiological effects of Ang II are produced through the activation of the AT1 receptor, which belongs to the G protein-coupled receptor (GPCR) superfamily. The AT1 receptor interacts with the G protein Gq/11, which activates a phospholipase C, which in turn generates inositol 1,4,5-trisphosphate (InsP3) and diacylglycerol from the cleavage of phosphatidylinositol 4,5-bisphosphate (3, 4). InsP3 causes the release of Ca2+ from an intracellular store and diacylglycerol recruits and activates protein kinase C at the plasma membrane. As with all the hormones that trigger this signaling pathway, Ang II is recognized as a Ca2+-mobilizing hormone.

In the last decade, great emphasis has been placed on elucidating the various structural determinants involved in AT1 receptor activation at the molecular level. Like other GPCRs, the AT1 receptor undergoes spontaneous isomerization between its inactive state (favored in the absence of the agonist) and its active state (induced or stabilized by the agonist). With a photoaffinity labeling approach, we directly identified ligand-contact points within the second extracellular loop and the seventh transmembrane domain of the AT1 receptor (5, 6, 7). These contact points delimit the ligand-binding pocket of the receptor and may be important for its activation. Numerous mutagenesis studies have also provided indirect evidence for the involvement of transmembrane segments and/or specific amino acid side chains in agonist recognition and receptor activation (reviewed in Refs. 1 and 8). Based on these studies, Joseph et al. (9) proposed a preliminary model postulating that an interaction between Asn111 in the third transmembrane domain and Tyr292 in the seventh transmembrane domain maintains the AT1 receptor in the inactive conformation. The agonist Ang II would disrupt this interaction, allowing Tyr292 to interact with Asp74 in the second transmembrane domain and promote an active conformation. These authors later validated their model in part by showing that substituting Asn111 with Ala produces a constitutively active mutant receptor that signals in an agonist-independent fashion (10). Almost simultaneously, two other studies reported that the substitution of Asn111 with Ala produces a constitutively active AT1 receptor (11, 12). Feng et al. (13) further showed that a reduction of the side chain size of residue 111 induces an intermediate active conformation (N111G-AT1 being the most active mutant). Constitutively active mutant AT1 receptors obtained by replacing Asn111 have high affinities for agonist ligands and also exhibit increased efficacies in response to partial agonist and even some antagonist ligands (10, 11, 12, 13, 14, 15, 16). It is unclear whether the constitutive activity is due to a conformational state conferring a more efficient coupling to the G protein or to a conformational state that resembles the agonist-occupied receptor.

To clarify this question, we produced constitutively active mutant AT1 receptors by substituting Asn111 with smaller amino acid residues (Ala or Gly) and we also obtained less activatable mutant receptors by substituting Asn111 with larger amino acid residues (Gln or Trp). The pharmacological and functional properties of these receptors were analyzed after transient expression in COS-7 cells. Their coupling properties were indirectly assessed by binding studies in the presence of uncoupling agents and directly assessed in receptor/G protein coimmunoprecipitation studies. We showed that the less activatable mutant receptors poorly couple whereas the constitutively active mutant receptors efficiently and reversibly couple to Gq/11{alpha}. More importantly, we showed that the constitutively active N111G-AT1 receptor maintains a high affinity conformation despite its uncoupling from Gq/11.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
The cDNA encoding the human AT1 receptor with a N terminus FLAG epitope was constructed in our laboratory and subcloned in the mammalian expression vector pcDNA3 (Invitrogen, San Diego, CA). The Sculptor in vitro mutagenesis kit, restriction endonucleases, polymerases, myo-[3H]-inositol (80 Ci/mmol), 125iodine (2000 Ci/mmol) and ECL plus Western blotting detection reagents were from Amersham Pharmacia Biotech (Piscataway, NJ). DMEM, fetal bovine serum (FBS), penicillin-streptomycin-glutamine, lipofectamine and oligonucleotide primers were from Life Technologies, Inc. (Gaithersburg, MD). COS-7 cells were from the American Type Culture Collection (Manassas, VA). Ang II, [Sar1, Ile8]Ang II, BSA, bacitracin, soybean trypsin inhibitor (STI), CNBr, monoclonal anti-FLAG M1 antibody, and FLAG peptide were from Sigma (Oakville, Ontario, Canada). The AG 1-X8 resin was from Bio-Rad (Mississauga, Ontario, Canada). The protease inhibitors aprotinin, leupeptin and pefabloc SC were from Roche (Mannheim, Germany). Goat polyclonal antirabbit-IgG antibody conjugated to horseradish peroxidase, rabbit polyclonal anti-Gq/11{alpha} antibody (C-19) and protein A/G plus-agarose beads were from Santa Cruz Biotechnology (Santa Cruz, CA). Immobilon-P polyvinylidene fluoride (PVDF) transfer membranes were from Millipore (Bedford, MA). [Sar1, Bpa8]Ang II was synthesized in our laboratory by the solid-phase method and purified by HPLC as previously described (17). 125I-Ang II, 125I-[Sar1, Ile8]Ang II and 125I-[Sar1, Bpa8]Ang II (1000 Ci/mmol) were prepared with IODO-GEN (Pierce, Rockford, IL) according to the method of Fraker and Speck (18) in an acetic acid buffer (pH 5.4) and purified by HPLC on a C-18 column (Waters, Mississauga, Ontario, Canada) as previously reported (19). The specific radioactivities of the radiolabeled peptides were determined by self-displacement and saturation binding experiments as described by Boulay et al. (20).

Construction of the mutant receptors
The cDNA encoding the human AT1 receptor was inserted into HindIII and XbaI sites of M13mp19. Site-directed mutagenesis was done using the Sculptor in vitro mutagenesis kit. Four oligonucleotides were constructed to introduce different mutations at Asn111. The oligonucleotide primers were the following (altered nucleotides are underlined): Asn111-> Gly (N111G-AT1), 5'-GCGTACAGGCCGAAACTGACG-3'; Asn111-> Ala (N111A-AT1), 5'-CTAGCGTACAGGGCGAAACTGACGCT-3'; Asn111-> Gln (N111Q-AT1), 5'-GCTAGCGTACAGCTGGAAACTGACGC-3'; Asn111-> Trp (N111W-AT1), 5'-GCTAGCGTACAGCCAGAAACTGACGCT-3'. After confirmation of the site-directed mutations and integrity of the cDNAs by DNA sequencing, the N111G-AT1, N111A-AT1, N111Q-AT1 and N111W-AT1 cDNAs were excised from the M13mp19RF by digestion with HindIII and XbaI and subcloned into the multiple cloning site of pcDNA3 digested with the same restriction enzymes. The FLAG epitope was inserted in frame with the coding sequence of the different constructs by a subcloning strategy using the restriction endonucleases AccI, HindIII, and XbaI.

Cell culture and transfection
COS-7 cells were grown in DMEM supplemented with 10% [vol/vol] heat-inactivated FBS, 2 mM L-glutamine, 100 IU/ml penicillin, and 100 µg/ml streptomycin (complete DMEM). A total of 1 x 106 cells were seeded into 75-cm2 culture dishes. After 24 h of growth, the cells were washed once with serum-free DMEM and transfected with 4 µg of plasmid DNA and 25 µl of lipofectamine in 8 ml of serum-free DMEM. The cells were incubated for 5 h at 37 C, and the medium was replaced with complete DMEM. The transfected cells were allowed to grow for 12 h, transferred to six-well culture plates for inositol phosphate (IP) production experiments or directly seeded on coverslips for Ca2+ mobilization studies. Cells were used 36–60 h after the initial transfection. For photoaffinity labeling, binding and coimmunoprecipitation assays, the cells were grown for 48 h in 75-cm2 culture dishes and stored at -80 C.

Binding experiments
Broken cells (frozen and thawed) were gently scraped into 10 ml of washing buffer [25 mM Tris-HCl (pH 7.4), 100 mM NaCl, and 5 mM MgCl2] and centrifuged at 2500 x g for 15 min at 4 C. The pellet was dispersed in binding buffer [25 mM Tris-HCl (pH 7.4), 100 mM NaCl, 5 mM MgCl2, 0.1% (wt/vol) BSA, 0.01% (wt/vol) bacitracin, and 0.01% (wt/vol) STI]. Saturation binding studies were performed by incubating broken cell aliquots (20–50 µg of protein) for 1 h at room temperature in a final volume of 0.5 ml of binding buffer containing varying concentrations of radioactive and nonradioactive ligands. Nonspecific binding was measured in the presence of 1 µM unlabeled Ang II. Bound radioactivity was separated from free ligand by vacuum filtration through glass microfiber filters of grade 691 (VWR International, West Chester, PA) presoaked for 2 h in binding buffer. Receptor-bound radioactivity was evaluated by {gamma} counting. Binding affinities [dissociation constant (Kd)] and receptor expression levels [maximal binding capacity (Bmax)] were calculated by Scatchard analysis of the saturation curves.

Photoaffinity labeling
Photoaffinity labeling experiments were essentially done as previously described (21). Briefly, broken cells aliquots were incubated for 1 h at room temperature in the presence 5 nM 125I-[Bpa8]Ang II in 0.5 ml of binding buffer. After washing by centrifugation at 500 x g for 15 min, the broken cells were resuspended in 0.5 ml of ice-cold binding buffer (without BSA) and irradiated for 1 h at 0 C under filtered UV light (365 nm). Broken cells were then solubilized in RIPA buffer [50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 0.5% (wt/vol) deoxycholate, 0.1% (wt/vol) sodium dodecyl sulfate (SDS), and 1% (vol/vol) Nonidet P-40]. After centrifugation for 15 min at 15,000 x g, the supernatant was mixed with an equal volume of 2x Laemmli buffer [60 mM Tris-HCl (pH 6.8), 10% (vol/vol) glycerol, 2% (wt/vol) SDS, 100 mM dithiothreitol, and 0.05% (wt/vol) bromophenol blue], incubated for 1 h at 37 C and analyzed by SDS-PAGE on a 7.5% (wt/vol) polyacrylamide Tris-glycine gel.

Chemical digestion
Partially purified photolabeled receptors (5,000–10,000 cpm) were incubated in a mixture containing 200 µl of 70% (vol/vol) trifluoroacetic acid and 200 µl of CNBr to obtain a final concentration of 100 mg/ml. Samples were incubated for 18 h at room temperature in the dark. Reactions were terminated by the addition of 1 ml of water. After lyophilization and resuspension in 1x Laemmli buffer, samples were analyzed by SDS-PAGE on 16.5% (wt/vol) polyacrylamide Tris-tricine gels and revealed by autoradiography on BioMax MS film (Eastman Kodak, Rochester, NY).

IP production
COS-7 cells were seeded into six-well plates 12 h post transfection, allowed to grow for 24 h and labeled for 16–24 h in inositol-free DMEM containing 10 µCi/ml of myo-[3H]-inositol. After preincubation for 30 min at 37 C in Medium 199 containing 25 mM HEPES (pH 7.4), 10 mM LiCl and 0.1% (wt/vol) BSA, cells were stimulated with 100 nM Ang II for 20 min. The incubation was stopped by adding PCA [5% (vol/vol)]. Water-soluble IPs were then extracted with an equal volume of a 1:1 mixture of 1,1,2-trichlorotrifluoroethane and tri-n-octylamine. The samples were vigorously mixed and centrifuged at 15,000 x g for 1 min. The upper phase was applied to an AG 1-X8 resin column and the IPs were sequentially eluted by the addition of an ammonium formate/formic acid solution of increasing ionic strength.

Measurement of intracellular Ca2+
Transfected COS-7 cells were grown on coverslips, washed twice with HBSS [20 mM HEPES (pH 7.4), 120 mM NaCl, 5.3 mM KCl, 0.8 mM MgSO4, 1.8 mM CaCl2, and 11.1 mM glucose] and loaded with Fura-2/AM (1 µM in HBSS, Molecular Probes, Eugene, OR) for 20 min at room temperature in the dark. After washing and incubating in fresh HBSS for 20 min at room temperature, the coverslips were inserted into a circular open-bottom chamber and placed on the stage of a Zeiss Axovert microscope fitted with an Attofluor Digital Imaging and Photometry System (Attofluor Inc., Rockville, MD). The system allows data acquisition from up to 99 user-defined variably sized regions of interest per field of view. From 40–99 isolated Fura-2-loaded cells were selected and the [Ca2+]i was measured by fluorescence videomicroscopy at room temperature using alternating excitation wavelengths of 334 and 380 nm and measuring emitted fluorescence at 520 nm. The data are expressed as a ratio of Fura-2 fluorescence (334/380). Data from 20–40 individual cells that responded to Ang II were collected from each coverslip.

Coimmunoprecipitation studies
Broken cells (800 µg/500 µl of binding buffer) were added to an equal volume of 2 x solubilizing buffer (binding buffer with 0.4% (wt/vol) CHAPS, 0.4 mM Pefabloc SC, 20 µM leupeptin, 10 µg/ml aprotinin, and 1 mM CaCl2) and incubated for 30 min at 4 C with gentle agitation. After centrifugation at 20,000 x g for 30 min at 4 C, the supernatant was transferred to 15 µl of wet protein A/G plus-agarose beads that had been preincubated with 4 µl of anti-FLAG M1 antibody(10 µg/ml). Immune complex formation was allowed to proceed for 3 h at 4 C with rotation. The agarose beads were sedimented by centrifugation at 5000 x g for 3 min and washed three times with ice-cold 1x solubilizing buffer. Beads were resuspended in 35 µl of loading buffer [60 mM Tris-HCl (pH 6.8), 10% (vol/vol) glycerol, 3% (wt/vol) SDS, 5% (vol/vol) ß-mercaptoethanol, and 0.05% (wt/vol) bromophenol blue] and incubated for 1 h at 50 C. After centrifugation at 5000 x g for 3 min, proteins from the supernatant were separated by SDS-PAGE on a 10% (wt/vol) polyacrylamide Tris-glycine gel and transferred to a PVDF membrane. The membrane was then blocked in Tris-buffered saline [20 mM Tris-HCl (pH 7.6) and 200 mM NaCl] supplemented with 5% (wt/vol) nonfat dry milk and 0.1% (vol/vol) Tween 20. The Gq/11{alpha} protein was probed with rabbit polyclonal anti-Gq/11{alpha} antibody (C-19) and goat polyclonal antirabbit-IgG antibody conjugated to horseradish peroxidase. The immunostained bands were revealed by enhanced chemiluminescence according to the manufacturer’s instructions on a BioMax ML film. Autoradiograms of membranes were digitized on a Hewlett Packard Scan Jet 5100c. Integrated peak areas were determined using the gel analysis Quantity One software (version 4.2; Bio-Rad).

Data analysis
Results are presented as the mean ± SD. Binding data (Bmax and Kd values) were analyzed with the Kell program (Biosoft, Ferguson, MO), which uses a weighted nonlinear curve-fitting routine.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Binding properties of mutant AT1 receptors
cDNAs encoding wild-type and mutant human AT1 receptors were subcloned into the pcDNA3 mammalian expression vector and transfected into COS-7 cells. The pharmacological properties of the different receptors were assessed in saturation binding studies with the radioactive agonist 125I-Ang II. As summarized in Table 1Go, the wild-type AT1 and the N111Q-AT1 receptors displayed two affinity states, suggesting that they could adopt a high affinity (G protein coupled) conformation and a low affinity (G protein uncoupled) conformation. The N111W-AT1 receptor displayed a single low affinity state (Kd of 2.7 nM), whereas the N111G-AT1 receptor displayed a single high affinity state (Kd of 0.7 nM). Interestingly, in competitive binding assays, the N111G-AT1 receptor exhibited a relatively lower affinity than the WT-AT1 receptor for the nonpeptide antagonist losartan (data not shown). The level of expression of the different receptors varied between 1.7 and 2.7 pmol/mg of protein (Table 1Go).


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  		TABLE 1. Binding properties of wild-type and mutant AT1 receptors

 
Covalent labeling of mutant AT1 receptors
In photoaffinity labeling experiments, the photosensitive analog 125I-[Bpa8]Ang II specifically labeled the wild-type and mutant AT1 receptors that migrated as glycoproteins with a typical broad band pattern between 60 and 130 kDa (Fig. 1AGo). To further characterize the ligand binding site, the photolabeled receptors were treated with CNBr, which cleaves on the C-terminal side of methionine residues (Fig. 1BGo). This chemical digestion of the wild-type receptor (Fig. 1BGo) produced two typical fragments migrating as sharp bands with apparent molecular masses of 6.9 and 9.4 kDa as previously reported (5, 6). CNBr digestion of the mutant AT1 receptors also produced the same typical fragments (Fig. 1BGo), indicating that there were no major differences between the conformational states adopted by the different receptors in the presence of the photosensitive ligand.



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  		FIG. 1. Photoaffinity labeling of wild-type and mutant AT1 receptors. COS-7 cells expressing the wild-type AT1 receptor (WT) and various mutant receptors (N111G, N111A, N111Q, N111W) were incubated in the presence of 5 nM 125I-[Bpa8]Ang II for 1 h at room temperature. Cells were then irradiated under 365 nm filtered UV light for 1 h at 0 C. A, After solubilization, samples were resolved by SDS-PAGE on a 7.5% (wt/vol) polyacrylamide Tris-glycine gel followed by autoradiography as described in the experimental procedures. B, CNBr (100 mg/ml) hydrolysis of partially purified 125I-[Sar1, Bpa8]Ang II-labeled receptors proceeded for 18 h at room temperature in the dark before resolution by SDS-PAGE. Protein standards with the indicated molecular masses were run in parallel. These results are representative of three independent experiments.

 
Functional properties of mutant AT1 receptors
The functional properties of wild-type and mutant AT1 receptors were evaluated by assessing the basal and Ang II-induced production of IPs in transiently transfected COS-7 cells. Figure 2Go shows the relative amounts of IPs accumulated under basal conditions (white columns) and after maximal stimulation with Ang II (black columns). The basal levels of IPs in cells expressing the mutant N111Q-AT1 and N111W-AT1 receptors were relatively low and not significantly different from the basal level in cells expressing the wild-type AT1 receptor. As expected, the basal levels of IPs in cells expressing the mutant N111A-AT1 and N111G-AT1 receptors were significantly higher than those in cells expressing the wild-type AT1 receptor. These results illustrate the constitutive activity of the mutant N111A-AT1 and N111G-AT1 receptors. After maximal stimulation with Ang II, the constitutively active receptors increased the IPs to levels not significantly different from those of the wild-type AT1 receptor. Interestingly, after maximal stimulation, the N111Q-AT1 receptor caused only a weak production of IPs, whereas the N111W-AT1 receptor did not significantly elevate the level of IPs. The mutant N111Q-AT1 and N111W-AT1 receptors are clearly less activatable than the wild-type and the constitutively active mutant AT1 receptors. These results provide a strong support to the previous suggestion by Feng et al. (13) that an increase in the side chain size of residue 111 leads to a form of the receptor with reduced basal and maximal activities.



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  		FIG. 2. Functional properties of the wild-type and mutant AT1 receptors: IP production. Transfected COS-7 cells were loaded for 16–24 h with 10 µCi/ml of myo-[3H]-inositol in inositol-free DMEM. Cells were then incubated for 20 min in the presence (black columns) or absence (white columns) of 100 nM Ang II and IP levels (sum of InsP2 and InsP3) were determined as described in the experimental procedures. These results represent the means ± SDs of at least three experiments (done in triplicate) where IP production was normalized for receptor expression level (determined by saturation binding assays). *, P < 0.05 compared with basal value for WT-AT1; {dagger}, P < 0.05 compared with Ang II-stimulated WT-AT1.

 
The functional properties of the different receptors were also evaluated with a Fura-2 fluorescence approach that measures the free calcium concentration within individual living cells. Addition of Ang II (100 nM) to COS-7 cells transfected with the wild-type AT1 receptor resulted in a rapid, robust increase in intracellular Ca2+, which reached a maximum level within a few seconds and then declined toward a lower level that remained slightly above the resting level for at least 3 min (Fig. 3AGo). Under similar conditions, the N111W-AT1 receptor caused a much weaker Ca2+ transient that also declined to a level slightly above the resting level (Fig. 3AGo). Although we could not measure any significant increase of IPs upon stimulation of this receptor, these results demonstrated that it can nevertheless couple to Gq/11{alpha} with a low efficacy and produce enough InsP3 to cause a weak Ca2+ transient. Surprisingly, maximal stimulation of the N111G-AT1 receptor consistently produced a Ca2+ transient that had a lower amplitude than that produced by the wild-type receptor and that declined to the resting level within a few minutes (Fig. 3AGo). Because this constitutively active receptor was shown to be as efficient as the wild-type receptor in producing IPs, these results suggest that the cells expressing the N111G-AT1 receptor have developed a refractoriness in their Ca2+ mobilization mechanism at a step downstream from phospholipase C. A possible cause for this reduced response could be the level of Ca2+ within the intracellular stores that could possibly be maintained lower in cells containing higher basal InsP3 concentrations. This possibility is unlikely, however, because in a nominally free extracellular Ca2+ medium, thapsigargin (a SERCA inhibitor) released the same amount of Ca2+ from cells expressing the N111G-AT1 receptor as from cells expressing the WT-AT1 receptor (Fig. 3BGo).



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  		FIG. 3. Functional properties of the wild-type and mutant AT1 receptors: Ca2+ mobilization. A, Transfected COS-7 cells were loaded with Fura-2/AM and their [Ca2+]i was monitored upon stimulation with 100 nM Ang II (filled arrow). Typical traces represent the average Ang II-induced (100 nM) Ca2+ transients in 150–350 cells transfected with the wild-type AT1 receptor (filled circle), the mutant N111G-AT1 receptor (empty circle), the mutant N111W-AT1 receptor (filled square), and the empty pcDNA3 plasmid (empty square). B, QBI 293A cells stably expressing the WT-AT1 receptor (WT) or the N111G-AT1 receptor (N111G) were loaded with Fura-2/AM and their Ca2+ stores content was evaluated by addition of thapsigargin (1 µM; empty arrow) in a nominally free extracellular Ca2+ medium. These typical traces summarize the results of four independent experiments.

 
G protein coupling of mutant AT1 receptors
We used a classical binding approach to assess the G protein coupling efficacies of the mutant AT1 receptors in the presence of an uncoupling agent. Figure 4AGo shows a typical dose-displacement experiment where the binding of 125I-Ang II to the wild-type AT1 receptor was proportionally decreased in the presence of increasing concentrations of nonradioactive Ang II. Under control conditions (white diamond), the tracer bound with a high affinity (15,700 cpm specifically bound) and the concentration of nonradioactive Ang II required to inhibit 50% of tracer binding (IC50) was 0.8 nM. In the presence of the uncoupling agent GTP{gamma} S (black square), the tracer bound with a lower affinity (3600 cpm specifically bound) and the IC50 of Ang II was increased by about 3-fold (2.1 nM). This typical experiment clearly illustrated the loss of affinity of the AT1 receptor for its agonist ligand upon treatment with an uncoupling agent.



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  		FIG. 4. Binding properties in the presence of an uncoupling agent. Broken cells (20–50 µg of protein) expressing the wild-type AT1 receptor (A), the N111W-AT1 receptor (B), and the N111G-AT1 receptor (C) were incubated for 1 h at room temperature in binding buffer containing 0.1 nM 125I-Ang II and increasing concentrations of Ang II, in the absence (empty symbols) or presence of 10 µM GTP{gamma} S (filled symbols). D, Broken cells expressing the N111G-AT1 receptor were preincubated for 1 h in the presence of 0.1 nM 125I-Ang II and then incubated in the presence (empty symbols) or absence (filled symbols) of the uncoupling agent for different periods of time. Incubations were terminated by vacuum filtration as described in the experimental procedures. Each point represents the mean ± experimental variation of duplicate data. Similar results were obtained with three different cell preparations.

 
The same experiment was repeated with the less activatable mutant N111W-AT1 receptor (Fig. 4BGo). Interestingly, under control conditions (white diamond), the tracer bound with a low affinity (3000 cpm specifically bound) and the IC50 of Ang II was 5.1 nM. In the presence of the uncoupling agent (black square), the dose-displacement curve was superimposable on the control curve. These results suggest that the mutant N111W-AT1 receptor is not basally coupled to its cognate G protein. This interpretation is consistent with the relatively low affinity of this mutant for the agonist ligand, with its poor activation of phospholipase C and with its induction of a weak Ca2+ transient.

These experiments were repeated with the constitutively active N111G-AT1 receptor. Figure 4CGo shows that under control conditions (white diamond), the tracer bound with a high affinity (21,300 cpm specifically bound) and the IC50 of Ang II was 1.8 nM. In the presence of the uncoupling agent (black square), the dose-displacement curve was superimposable on the control curve. The naïve interpretation of these results would be that this receptor does not couple to a G protein because it is insensitive to the effect of the uncoupling agent. However, this interpretation is unlikely considering that the mutant N111G-AT1 receptor is a strong activator of phospholipase C, most probably through the activation of the G protein Gq/11. One possibility could be that the coupling between the constitutively active receptor and its G protein is so strong that it requires a longer period of treatment with the uncoupling agent to dissociate the two proteins. Figure 4DGo shows that the binding of 125I-Ang II to the constitutively active receptor was not modified during incubation with an uncoupling agent for periods as long as 5 h. These results suggest a very strong and virtually undissociable coupling between the constitutively active receptor and its G protein. An alternative explanation for these results could be that the receptor always remains in a high agonist affinity conformation, whether or not it is coupled to its G protein.

To discriminate between these two possibilities, we used a coimmunoprecipitation approach to evaluate the coupling state of the mutant AT1 receptors in a more direct fashion. Figure 5Go shows that under basal conditions, immunoprecipitation of the wild-type AT1 receptor coprecipitated a small amount of the G protein Gq/11{alpha} (panel A). The coprecipitation of Gq/11{alpha} increased upon stimulation with Ang II and decreased in the presence of the uncoupling agent GTP{gamma} S. The coimmunoprecipitation was specific and did not occur when the anti-FLAG antibody was previously blocked with a saturating amount of FLAG peptide (panel B). Interestingly, when the constitutively active N111G-AT1 receptor was immunoprecipitated under basal conditions, only a small amount of Gq/11{alpha} was coprecipitated (panel A). Stimulation with Ang II strongly stabilized the complex between the constitutively active receptor and Gq/11{alpha}, whereas the uncoupling agent very efficiently destabilized this complex. With this approach, the less activatable N111W-AT1 receptor appeared poorly coupled to Gq/11{alpha}, under basal conditions and in the presence of Ang II (panel A). Panel C shows the densitometric analysis of results from panel A. These results demonstrate that the constitutively active AT1 receptor reversibly couples to the G protein Gq/11{alpha} and that the coupling is stabilized in the presence of the agonist Ang II and efficiently destabilized in the presence of GTP{gamma} S.



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  		FIG. 5. Coimmunoprecipitation of Gq/11{alpha} with the different AT1 receptors. COS-7 cells expressing the FLAG-AT1 receptor (WT), the FLAG-N111G-AT1 receptor (N111G) or the FLAG-N111W-AT1 receptor (N111W) were incubated for 1 h at room temperature in the presence or in the absence of 100 nM Ang II and/or 10 µM GTP{gamma} S. After solubilization with 0.4% CHAPS, FLAG-tagged receptors were immunoprecipitated with the anti-FLAG M1 antibody and the presence of Gq/11 in the immune complex was revealed by Western blot with the anti-Gq/11{alpha} antibody (C-19). A, Lanes 1–4, coimmunoprecipitation of Gq/11{alpha} under different conditions. As controls, a standard Gq{alpha} from E. coli (lane 5, empty arrow) and Gq/11{alpha} from COS-7 cells (lane 6, filled arrow) were run in parallel. B, The presence of the FLAG-WT-AT1 receptor in the immune complex under the different conditions was revealed by Western blot with the anti-FLAG M1 antibody. C, Densitometric analysis of the results shown in panel A. These results are representative of three experiments performed with different cell preparations.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Three independent studies previously suggested that Asn111 plays a conformational switch function in the activation of the AT1 receptor (10, 11, 12). Another recent study demonstrated that only residues smaller than Asn can confer constitutive activity on the AT1 receptor and therefore concluded that a feature responsible for constitutive activity of the AT1 receptor is the side chain size of the residue at position 111 (13).

In the study presented here, we evaluated the pharmacological and functional properties of mutant AT1 receptors in which Asn111 was replaced by different residues of various sizes, including the smaller Gly and larger Trp natural amino acid residues. In transient transfection studies, all the mutant receptors were expressed at the same high levels as that of the wild-type receptor (1–2 pmol/mg protein). In photoaffinity labeling experiments, identical CNBr fragmentation patterns revealed that the mutant receptors were covalently labeled at the same location as the wild-type receptor. Except for the N111W mutant, which exhibited a 4-fold reduction in binding affinity, all of the mutant receptors recognized Ang II with a high affinity similar to that of the wild-type receptor. These results are in agreement with those of previous studies reporting minor reductions in binding affinity due to the substitution of Asn111 with the larger residues Phe and Lys (11, 13). As also previously reported in other studies (16, 22), we observed that the substitution of Asn111 with smaller residues (Ala, Gly) caused a significant decrease in binding affinity for the nonpeptide antagonist losartan but no major changes for peptide agonists (data not shown). These results demonstrate that substitutions of Asn111 with smaller or larger amino acid residues caused only minor changes in the binding conformation of the AT1 receptor. These minor changes did not interfere with the proper folding of the receptor, which was correctly targeted and expressed at the plasma membrane and which maintained the same interaction with the photosensitive analog of Ang II.

The entire spectrum of functional states could be obtained by replacing the Asn111 of the AT1 receptor with different amino acid residues of increasing sizes. Substitutions with smaller residues (Gly, AL) produced constitutively active receptors. The smallest residue at position 111 produced the strongest constitutive activity. Substitutions with larger residues (Gln, Trp) produced less activatable receptors. The largest residue at position 111 produced an apparently inactive receptor with respect to the production of IPs. However, Ca2+ mobilization, a much more sensitive functional assay, revealed that all the mutant receptors, including the less activatable N111W-AT1, could produce an intracellular Ca2+ transient in response to Ang II. These results suggest that, despite its weak activity, the N111W mutant could still couple with a low efficacy to the G protein Gq/11 and induce the production of a sufficient amount of InsP3 to generate a detectable Ca2+ response. Another explanation could be that the intracellular Ca2+ transient obtained with the N111W mutant receptor is produced by a mechanism that does not requires the activation of a G protein. It was recently shown that a mutant AT1 receptor, lacking G protein coupling, was able to activate Src tyrosine kinase, possibly leading to transactivation of epidermal growth factor receptor and ultimately to activation of phospholipase C{gamma} (23).

Previous studies have shown that substituting Asn111 with smaller residues (Ala or Gly) conferred constitutive activity to the AT1 receptor (10, 11, 12, 13, 14, 15, 16). Noda et al. (11) further observed that N111I and N111F mutant receptors had lower basal activities and lower Ang II-stimulated maximal activities. These results led them to propose that Asn111 plays a crucial role in constraining the AT1 receptor in a basal inactive conformation. Substitution of Asn111 with a smaller residue would provide more flexibility to the receptor and favor acquisition of the active conformation. In contrast, substitution of Asn111 with a larger residue would further constrain the receptor in an inactive conformation, thus reducing its basal and maximal (agonist-induced) activities. This interpretation is very compatible with the model proposed by Groblewski et al. (10), who suggested that Asn111 restrains the AT1 receptor in an inactive conformation by forming an intramolecular bond with Tyr292. During the activation process of the AT1 receptor, the agonist would disrupt this intramolecular interaction and promote conformational flexibility. In the study presented here, we substituted Asn111 with Trp, the largest natural amino acid, and observed that the mutant receptor was barely activatable. Our results thus strongly support the current models on the molecular activation mechanism of the AT1 receptor.

The first mechanistic event occurring after activation of a GPCR is the recruitment and activation of its cognate G protein. Mutations that influence the functionality of the AT1 receptor may also affect its G protein coupling properties. As expected, we observed that the agonist-binding affinity of the wild-type AT1 receptor was decreased in the presence of the uncoupling agent GTP{gamma} S. This well-known phenomenon illustrates the efficient coupling and uncoupling capacities of the receptor (24, 25). Not surprisingly, the agonist-binding affinity of the N111W mutant was not modified in the presence of GTP{gamma} S, suggesting that this mutant couples poorly to Gq/11. This is consistent with the 4-fold lower agonist-binding affinity of this mutant compared with that of the wild-type receptor (Table 1Go). Interestingly, as previously observed by Noda et al. (11), the agonist-binding affinity of the constitutively active N111G mutant was not modified in the presence of GTP{gamma} S. This result could mean that the constitutively active receptor does not couple to Gq/11. However, the high efficiency of this receptor for the production of IPs argues against this interpretation. Two possibilities could therefore explain the lack of effect of GTP{gamma} S on the binding affinity of the N111G mutant. Either the interaction between the receptor and Gq/11 is so strong that GTP{gamma} S cannot dissociate both proteins, or the receptor maintains a high affinity conformation despite its dissociation from Gq/11.

Our coimmunoprecipitation approach revealed that under basal conditions the AT1 receptor was not strongly coupled to its cognate G protein Gq/11{alpha}. However, the agonist Ang II stabilized the complex between the two proteins whereas GTP{gamma} S destabilized it. To our knowledge, these are the first results showing the agonist-dependent coimmunoprecipitation of the AT1 receptor with Gq/11{alpha}. These results are consistent with those of previous studies that demonstrated the agonist-dependent coimmunoprecipitation of the cholecystokinin receptor with Gq/11{alpha} (26), the {delta} opioid receptor with Gi{alpha} (27), the IL-8 receptor with Gi{alpha} (28), and the melatonin receptor with Gi{alpha} and Gq/11{alpha} (29).

Not surprisingly, the N111W mutant did not coimmunoprecipitate a detectable amount of Gq/11{alpha} either under basal conditions or after stimulation with Ang II. Interestingly, the constitutively active N111G mutant behaved like the wild-type receptor with respect to coupling to Gq/11{alpha}. Because the N111G mutant could induce a relatively significant production of IPs (about 40% of maximal production) in the absence of Ang II, indicating a functional interaction with its cognate G protein, it was expected that it could coimmunoprecipitate Gq/11{alpha}. However, it appears that under basal conditions the coupling of this mutant receptor to Gq/11{alpha} is rather weak or unstable and that Ang II is required to strengthen the complex. Ang II is probably stabilizing the receptor in a conformational state propitious for a strong and efficient interaction with Gq/11{alpha}. This interpretation is consistent with the suggestion by Noda et al. (10) that a decrease in the size of the Asn111 side chain induces an intermediate-activated receptor conformation (R') that can isomerize to the fully activated conformation (R*) either spontaneously or after induction by Ang II. Because the physical coupling between the N111G mutant and Gq/11{alpha} was relatively low in the absence of agonist, it is likely that the spontaneous isomerization from the R' to the R* conformation occurs only transiently and that the agonist is necessary to stabilize it. Our results further showed that the N111G-AT1 receptor maintains a high agonist-binding affinity in the presence of GTP{gamma} S despite being completely uncoupled from Gq/11{alpha}. These results lend credence to the hypothesis by Kjelsberg et al. (30) that the high affinity state of constitutively active mutants of {alpha}1B-adrenergic receptors does not require an interaction with a G protein but is rather an intrinsic property of the receptors themselves.

What would be the physiological consequence of the occurrence of a N111G-AT1 mutant in a living organism? The N111G-AT1 receptor adopts a high agonist-binding affinity state that appears to correspond to an intermediate activated receptor conformation. In the absence of AngII, this receptor can induce 40% of maximal phospholipase C activation. However, under normal physiological conditions where cells are constantly exposed to low levels of Ang II, the high affinity of this receptor would promote the formation of a large number of functional ternary agonist receptor-G protein complexes and therefore cause very significant activation (probably much more than 40% of maximal level) of the intracellular mechanisms regulated by the AT1 receptor. Our Ca2+ mobilization studies revealed some refractoriness in cells expressing the N111G-AT1 receptor, suggesting that a desensitization process has developed as a consequence of the permanent activity of this receptor. We showed that this reduced response was not due to a reduced content of Ca2+ within the intracellular stores of cells expressing the N111G-AT1 receptor. Tovey et al. (31) reported that prolonged stimulation of Hela and SH-SY5Y neuroblastoma cells reduced the amplitude, duration and frequency of Ca2+ puff sites and showed that this effect was unlikely to be due to InsP3 production but rather to IP3R down-regulation, under these conditions. Further work is needed to identify the exact refractory component(s) of the Ca2+ cascade and also the phenotypic and mechanistic changes occurring in cells expressing the constitutively active N111G-AT1 receptor.

In conclusion, we have shown that the constitutively active N111G-AT1 receptor behaves similarly to the wild-type AT1 receptor as regard to G protein coupling but adopts a high agonist affinity conformation that is maintained in spite of its uncoupling from Gq/11{alpha}.


   Footnotes
 
This work is part of the Ph.D. thesis of M.A.M. and was supported by grants from the Canadian Institutes of Health Research. R.L. is a Scholar of the Fonds de la Recherche en Santé du Québec (FRSQ). E.E. is a recipient of a J.C. Edwards Chair in cardiovascular research. P.C.L. is a recipient of studentships from the Natural Sciences and Engineering Research Council of Canada (NSERC). M.A.M. and P.M.L. are recipients of studentships from FRSQ.

Abbreviations: Ang II, Angiotensin II; AT1 receptor, angiotensin II type 1 receptor; Bmax, maximal binding capacity; Bpa, p-Benzoyl-l-phenylalanine; CNBr, cyanogen bromide; ECL, enhanced chemiluminescence; FBS, fetal bovine serum; GPCR, G protein-coupled receptor; InsP2, inositol bisphosphate; InsP3, inositol trisphosphate; IP, inositol phosphate; Kd, dissociation constant; PVDF, polyvinylidene fluoride; RIPA, radioimmunoprecipitation assay; SDS, sodium dodecyl sulfate; SERCA, sarcoplasmic and endoplasmic reticulum calcium ATPase; STI, soybean trypsin inhibitor.

Received May 30, 2003.

Accepted for publication August 8, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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