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Endocrinology Vol. 143, No. 12 4702-4710
Copyright © 2002 by The Endocrine Society

ARTICLE

A Polyaromatic Caveolin-Binding-Like Motif in the Cytoplasmic Tail of the Type 1 Receptor for Angiotensin II Plays an Important Role in Receptor Trafficking and Signaling

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

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

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


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The type 1 receptor for angiotensin II (AT1) is a member of the G protein-coupled receptor family. The presence of a caveolin-binding-like motif ({phi}X{phi}XXXX{phi}XX{phi} where {phi} is an aromatic residue) within the cytoplasmic tail of the AT1 receptor suggests an implication for caveolae in the functionality of this receptor. We constructed a mutant AT1 receptor where each of the aromatic residues in the caveolin-binding-like motif were replaced by alanine (AT1-YFFY/A). Mutation of this motif considerably reduced the plasma membrane expression of the receptor that accumulated in a perinuclear compartment. The agonist-induced internalization rate of the AT1-YFFY/A receptor was also significantly reduced. Finally, the AT1-YFFY/A receptor was poorly activated as indicated by a low agonist-induced production of inositol phosphates. Unexpectedly, the proportion of AT1 receptor found in caveolae was minor under basal conditions and did not increase under stimulated conditions. Coexpression of the AT1 receptor with dopamine receptor interacting protein of 78 kDa, a protein implicated in the cellular routing of the dopamine D1 receptor, increased plasma membrane expression of the AT1 receptor. However, dopamine receptor interacting protein of 78 kDa had no effect on the expression of the AT1-YFFY/A receptor. Taken together, these results suggest that the caveolin-binding-like motif of the AT1 receptor does not promote localization of the receptor to caveolae but rather may act as a docking site for regulatory proteins modulating the routing and the functionality of the receptor.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE OCTAPEPTIDE HORMONE angiotensin II (Ang II) (1) produces a wide variety of physiological effects including vasoconstriction, aldosterone secretion, decreased glomerular filtration and cardiac myoproliferation (for review see Ref. 1). Although Ang II is known to interact with two receptor subtypes, designated AT1 and AT2, both belonging to the superfamily of G protein-coupled receptors (GPCRs) (2, 3, 4, 5, 6), the vast majority of its well-described effects are mediated by the AT1 receptor. The AT1 receptor is coupled to the G protein Gq, which leads to the activation of phospholipase C, thus generating the second messengers inositol 1,4,5-trisphosphate and diacylglycerol (7, 8, 9).

In an effort to better characterize the mechanism of action of the AT1 receptor, numerous studies have identified different domains or regions responsible for interaction with other proteins involved in its expression, its activation or its regulation. Among these regions, the seventh transmembrane domain was shown to directly interact with the agonist Ang II (10, 11), the DRY motif located in the N terminus of the second intracellular loop, the N terminus and the C terminus of the third intracellular loop, and the proximal region of the cytoplasmic tail were shown to be critical for coupling with Gq (12, 13). Moreover, the cytoplasmic tail was also shown to contain a binding domain for Ca2+/calmodulin (14) and possibly for ß-arrestin (15). Interestingly, the cytoplasmic tail of the AT1 receptor possesses an aromatic-rich sequence ({phi}X{phi}XXXX{phi}XX{phi}, where {phi} is an aromatic residue) with the specific spacing of the caveolin-binding motif identified by Couet et al. (16) using a phage display approach. Caveolin is the major protein component of caveolae, which are specialized regions of the plasma membrane enriched in sphingolipids, cholesterol, and lipid-anchored membrane proteins (17, 18, 19). Caveolae were first described as structures responsible for the uptake by potocytosis of small molecules such as folic acid, cholera toxin, and ions (20, 21, 22, 23). Many proteins, including adenylyl cyclase, nitric oxide synthase, trimeric G proteins, protein kinases, some GPCRs, and growth factors receptors, have been found in caveolae (24). It is now considered that caveolae serve as scaffolds to compartmentalize and integrate a wide range of signaling molecules forming preassembled signaling complexes.

The presence of a caveolin-binding-like motif Tyr302- X-Phe304-XXXX-Phe309-XX-Tyr312 in the cytoplasmic tail of the AT1 receptor (see Fig. 1Go) suggests that caveolae may play a role in the signaling mechanism of the AT1 receptor. In the study presented here, we used site-directed mutagenesis to disrupt this caveolin-binding-like motif. We discovered that this motif plays an important role in the targeting of the receptor to the plasma membrane, in the receptor activation of phospholipase C and in the agonist-induced receptor internalization. In spite of the importance of this motif on the functional properties of the AT1 receptor, we were unable to demonstrate any enrichment of the AT1 receptor in caveolae, under basal or agonist-stimulated conditions. We conclude that the aromatic motif is not promoting the association of the AT1 receptor with caveolin but possibly with other types of regulatory protein(s).



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  		Figure 1. Schematic representation of the human AT1 receptor. The AT1 receptor is part of the GPCR superfamily of proteins that possess seven membrane-spanning domains. Amino acid sequence of the caveolin-binding motif are depicted. Enlarged circles represent aromatic residues of the motif that were mutated for Ala. A FLAG epitope was inserted at the N terminus (filled circles) and N-glycosylation sites ({psi}) are represented.

 

   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
The cDNA clone encoding the human AT1 receptor with an N terminus FLAG epitope was constructed in our laboratory and subcloned in the mammalian expression vector pcDNA3 (Invitrogen, San Diego, CA). The cDNA clone encoding rat dopamine receptor interacting protein of 78 kDa (DRIP78) also in pcDNA3 was kindly provided by Dr. Q.Y. Zhou (University of California, Irvine, CA). DMEM, lipofectamine, fetal bovine serum (FBS), penicillin-streptomycin, and oligonucleotide primers were purchased from Life Technologies, Inc. (Gaithersburg, MD). The Complete protease inhibitor cocktail was purchased from Roche Molecular Biochemicals (Mannheim, Germany). The Sculptor in vitro mutagenesis kit, restriction endonucleases, polymerases, [3H]-myo-inositol (80 Ci/mmol), and 125Iodine (2000 Ci/mmol) were from Amersham Pharmacia Biotech (Piscataway, NJ). [125I]Ang II and [125I]-[Sar1, Ile 8]Ang II (1000 Ci/mmol) were prepared with Iodogen (Pierce Chemical Co., Rockford, IL) as previously described (25) and purified by HPLC on a C-18 column.

Construction of AT1-YFFY/A receptor and green fluorescent protein (GFP) fusion proteins
The pcDNA3 vector containing the human AT1 receptor cDNA was digested with HindIII and Xba1, and the resulting 1250-bp fragment was subcloned into the HindIII-Xba1-digested RF form of M13 mp19. The mutation of the caveolin-binding motif was achieved in two steps. A first mutation was performed using the oligonucleotide FFY/A 304–312 (5'-GAAGCTGGAGAAAAGCTCTTTTAGCTTTTTTCCCCAGAGCGCCATAAAAAAGAGG-3') (altered nucleotides are underlined). For a complete mutation of the caveolin-binding motif, a second round of mutagenesis with the oligonucleotide FFY/A -> Y302A (5'CCAGAGCGCCAGCAAAAAGAGG-3') was performed on the mutant obtained previously. The GFP fusion proteins (AT1-receptor-GFP and AT1-YFFY/A-receptor-GFP) were constructed by the PCR method using the T7 (5'-TAATACGACTCACTATAGGG-3') and the PCRBamAT1 (5'-TACAACGGATCCTCAACCTCAAAACATGGTG-3') oligonucleotides on pcDNA3-hAT1. To allow proper fusion of the two genes, PCRBamAT1 was designed to remove the stop codon of the receptor so that a BamHI restriction site could be inserted. The resulting DNA was digested with HindIII and BamHI restriction enzymes, and the 1250-bp amplified fragment was cloned into the HindIII-BamHI-digested pEGFP-N1 vector (CLONTECH Laboratories, Inc., Palo Alto, CA). All constructs were submitted to nucleotidic sequencing to confirm mutations and integrity of the cDNAs.

Cell culture and transfections
COS-7 cells were grown in DMEM supplemented with 10% heat-inactivated FBS, 50 IU/ml penicillin, and 50 µg/ml streptomycin. The day before transfection, 100-mm dishes were seeded with 106 cells, and six-well plates were seeded with 125,000 cells per well. Eighteen hours later, cells were washed and transfected in serum-free DMEM containing 4 µg of DNA and 25 µl of lipofectamine (in 100-mm dishes) or 0.5 µg of DNA and 4 µl of lipofectamine per well (in six-well plates). Cells were incubated for 5 h at 37 C, after which the medium was replaced with DMEM containing 10% FBS. Two days after transfection, cells were washed with PBS and used immediately for functional assays or stored at -80 C.

Binding experiments
Frozen cells were thawed, scraped in ice-cold washing buffer (25 mM Tris-HCl, pH 7.4; 100 mM NaCl; 5 mM MgCl2) and broken by five cycles of aspiration-expulsion with a 10-ml serological pipette tightly apposed to the bottom of the dish. Broken cells were centrifuged at 2500 x g for 15 min at 4 C and resuspended in binding buffer (25 mM Tris-HCl, pH 7.4; 100 mM NaCl; 5 mM MgCl2; 0.1% BSA; and 0.01% bacitracin). Broken cells (35 µg of protein) were incubated for 1 h at room temperature in binding buffer containing increasing concentrations of 125I-[Sar1-Ile8]Ang II in a final volume of 0.5 ml. Bound radioactivity was separated from free ligand by filtration through GF/C filters presoaked for 2 h in binding buffer. Nonspecific binding was measured in the presence of 1 µM unlabeled Ang II. Receptor-bound radioactivity was evaluated by {gamma} counting.

Confocal microscopy
Transiently transfected COS-7 cells were grown on 25 mm diameter glass coverslips (VWR Canlab, Ville Mont-Royal, Québec, Canada) in multiwell dishes. Cells were fixed with 4% paraformaldehyde (in PBS) for 30 min at room temperature and were examined with a scanning confocal microscope (NORAN Instruments, Inc., Middleton, WI) equipped with a krypton/argon laser and coupled to an inverted microscope with a 40x oil immersion objective (Nikon, Melville, NY). Specimens were excited at wavelength 488 nm. Emitted GFP fluorescence was measured at wavelength 509 nm. Optical sections were collected at 0.25-µm intervals with 10-µm pinhole aperture. Digitized images were obtained with 256 times line averaging and enhanced with INTERVISION software (NORAN Instruments Inc.) on Silicon Graphics O2-workstation.

[125I]-Ang II internalization assay
Internalization was evaluated as previously described (26). Briefly, transfected COS-7 cells were incubated for varying periods of time at 37 C in internalization buffer (25 mM HEPES, pH 7.4; DMEM, 0.1% BSA) containing 0.1 nM [125I]Ang II. Internalization was stopped by washing the cells three times with ice-cold PBS. Cells were then incubated for 10 min in 2 ml of ice-cold acidic solution (150 mM NaCl; 50 mM acetic acid, pH 3). The supernatant containing the acid-released radioactivity was collected for analysis. Cells were then solubilized with 0.1 N NaOH and their acid-resistant radioactive content was evaluated.

Phospholipase C assay
Transiently transfected COS-7 cells grown in six-well plates were prelabeled for 20 h in inositol-free DMEM containing 8 µCi/ml of myo-[3H]inositol. After two washes with PBS, cells were incubated for 30 min in the stimulation buffer (25 mM HEPES, pH 7.4; DMEM; 10 mM LiCl; and 0.1% BSA). Phospholipase C was activated with 100 nM Ang II for 45 min in stimulation buffer at 37 C. Incubations were terminated by the addition of ice-cold perchloric acid (5% vol/vol). Cells were scraped and centrifuged at 15,000 x g for 5 min. Water-soluble inositol phosphates were then extracted with an equal volume of the 1:1 mixture of 1,1,2-trichlorotrifluoroethane and tri-n-octylamine. The samples were vigorously mixed and centrifuged at 2500 x g for 10 min. The upper phase containing the inositol phosphates was applied to an AG1-X8 resin column. Inositol phosphates were sequentially eluted by addition of ammonium formate/formic acid mixtures of increasing ionic strength (27).

Measurement of [Ca2+]i
Two days after transfection, cells attached to coverslips were washed twice with Hanks’ balanced salt solution (HBSS) (120 mM NaCl; 5.3 mM KCl; 0.8 mM MgSO4; 1.8 mM CaCl2; 11.1 mM glucose; 20 mM HEPES, pH 7.4) and loaded with fura 2-acetoxymethylester (Molecular Probes, Inc., Eugene, OR; 2 µM in HBSS) for 20 min at room temperature in the dark. After washing and incubating in fresh HBSS for 20 min at room temperature to achieve de-esterification, the coverslips were inserted into a circular open-bottom chamber and placed onto the stage of a Zeiss Axovert microscope (Carl Zeiss, Jena, Germany) 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. Twenty to 30 isolated fura 2-loaded cells were selected, and [Ca2+]i in these cells was measured by fluorescence videomicroscopy at room temperature using alternating excitation wavelengths of 334 and 380 nm and monitoring emitted fluorescence at 520 nm. Free [Ca2+]i was calculated from 334/380 fluorescence ratios. Data from 5–15 cells per coverslip that have an Ang II-induced calcium release were collected per experiment.

Triton-based preparation of caveolae
Caveolae were purified according to the protocol of Lisanti et al. (28), with slight modifications. Briefly, COS-7 cells were transiently transfected with cDNAs encoding AT1-receptor-GFP or AT1-YFFY/A-receptor-GFP. At confluency, cells were lysed in an ice-cold 2-[N-morpholino]ethanesulfonic acid (MES)-buffered solution (MBS; 25 mM MES, pH 6.5; 150 mM NaCl) containing 1% Triton X-100, and protease inhibitors. After homogenization with 10 strokes of a Dounce homogenizer (Kontes Glass Co., Vineland, NJ), the extracts were adjusted to 45% sucrose with MBS (lacking Triton X-100), and distributed at the bottom of ultracentrifuge tubes. The samples were then overlaid with 4 ml of a 35% sucrose cushion followed by 4 ml of a 5% sucrose cushion, and centrifuged at 39,000 rpm for 16–20 h in an SW41 rotor (Beckman Instruments, Palo Alto, CA). Fractions (1 ml) were collected and their fluorescence intensity was monitored ({lambda}ex 488 nm, {lambda}em 509 nm) using a fluorescence spectrophotometer (F-2000, Hitachi, Hialeah, FL).

Detergent-free preparation of caveolae
This method was adapted from Song et al. (29). At confluency, COS-7 cells expressing AT1-receptor-GFP or AT1-YFFY/A-receptor-GFP were scraped in 2 ml of ice-cold 500 mM sodium carbonate, pH 11. Homogenization was carried out with three 10-sec bursts at maximum setting of a Polytron (Kinematica, Cincinnati, OH) tissue grinder. It was followed by one 30-sec burst at setting 4 and one 30-sec burst at setting 8 of a sonicator (Dismembrator 60, Fisher Scientific, Pittsburgh, PA). The homogenate was then adjusted to 45% sucrose by the addition of 2 ml of 90% sucrose prepared in MBS and placed at the bottom of an ultracentrifuge tube. The lysate was then overlaid with 4 ml of a 35% sucrose cushion and 4 ml of a 5% sucrose cushion, both prepared in MBS containing 250 mM sodium carbonate. The gradient was centrifuged at 39,000 rpm for 16–20 h in a SW41 rotor. Fractions (1 ml) were collected and their fluorescence intensity was evaluated.

Immunoblotting
Proteins from gradient fractions were precipitated with 5% perchloric acid, resuspended in Laemmli’s buffer, separated on a 12% SDS-polyacrylamide gel, and transferred to a nitrocellulose membrane (Amersham Pharmacia Biotech, Piscataway, NJ). After transfer, the membrane was blocked in Tris-buffered saline supplemented with 5% nonfat dry milk and 0.1% Tween 20 and subjected to immunoblotting with a monoclonal anticaveolin-1 antibody (Transduction Laboratories, Inc., Lexington, KY). Primary antibody was probed with an alkaline phosphatase-conjugated secondary antibody and visualized with the enhanced chemiluminescence system (Amersham Pharmacia Biotech) on a Bio-Max ML film (Eastman Kodak Co., Rochester, NY).

Data analysis
Results were collected in triplicate and presented as the mean ± SD. Binding curves, maximum binding capacity (Bmax) and dissociation constant (Kd) values were derived from 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 the AT1-YFFY/A receptor
We constructed a mutant AT1 receptor (AT1-YFFY/A) whose aromatic residues in the caveolin-binding-like motif were replaced by alanine (Fig. 1Go). The pharmacological properties of the AT1-YFFY/A receptor were assessed after transfection in COS-7 cells. Binding properties were evaluated with the antagonist ligand 125I-[Sar1, Ile8]Ang II. Figure 2AGo shows Scatchard representations of binding isotherms for the AT1 receptor (open circle) and the AT1-YFFY/A receptor (filled circle). Scatchard plot analysis yielded Kd values of 0.8 ± 0.2 nM and 2.8 ± 1.6 nM and Bmax values of 4.8 ± 2.1 pmol/mg of protein and 0.5 ± 0.3 pmol/mg of protein (three experiments) for the AT1 receptor and the AT1-YFFY/A receptor, respectively. These results demonstrate that the aromatic residues in the caveolin-binding-like domain of the cytoplasmic tail of the AT1 receptor are important for an optimal expression of the receptor at the plasma membrane but not for the recognition of the ligand [Sar1, Ile8]Ang II.



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  		Figure 2. Binding properties and cellular localization of AT1-YFFY/A receptor. A, Broken COS-7 cells (35 µg of protein) transiently transfected with the AT1 receptor ({circ}) or the AT1-YFFY/A ({bullet}) receptor cDNA were incubated for 1 h at room temperature with increasing concentrations of 125I-[Sar1,Ile8]Ang II. Bound and free 125I-[Sar1,Ile8]Ang II were separated by filtration through GF/C filter. Nonspecific binding was assessed in the presence of 1 µM unlabeled [Sar1,Ile8]Ang II. Each point represents the mean of triplicate values. Kd and Bmax values were evaluated by Scatchard analysis of the saturation curve data (representative of three independent experiments). COS-7 cells expressing the AT1-receptor-GFP (B) or the AT1-YFFY/A-receptor-GFP (C) were grown on circular coverslips (25-mm diameter), fixed with paraformaldehyde (4% in PBS) and observed by confocal microscopy. These images are representative of at least three independent experiments.

 
Cellular localization of the AT1-YFFY/A receptor
To determine the cellular localization of the receptors, we constructed fusion proteins where GFP was fused to the C termini of native and mutant receptors. We previously showed that the presence of the GFP protein did not alter the pharmacological or functional properties of the AT1 receptor (30). Confocal fluorescence microscopy of COS-7 cells expressing the AT1-receptor-GFP revealed a strong labeling of the plasma membrane and a weak labeling within the cytoplasm (Fig. 2BGo). In cells expressing the AT1-YFFY/A-receptor-GFP, the fluorescence was mostly distributed to the perinuclear region and in very low proportion to the plasma membrane, which was barely discernible (Fig. 2CGo). These results show that the mutant receptor accumulates intracellularly suggesting that the caveolin-binding-like motif is implicated in the proper routing of the AT1 receptor to the plasma membrane.

Functional properties of the AT1-YFFY/A receptor
To evaluate the contribution of the caveolin-binding-like motif in receptor internalization, we assessed the kinetics of 125I-Ang II internalization. Figure 3AGo shows the time course of internalization of 125I-Ang II in COS-7 cells expressing the AT1 receptor (open circle) and the AT1-YFFY/A receptor (filled circle). During incubation at 37 C, the acid-resistant binding of 125I-Ang II to AT1 receptor increased rapidly with an initial rate of internalization of 9 ± 1.5%/min to reach a high steady-state value of 71 ± 3% within 30 min. This result is consistent with those reported in previous studies on AT1 receptor internalization (26, 31). The AT1-YFFY/A receptor had a slow rate of internalization (1.5 ± 0.2%/min) and reached a high steady-state value of 43 ± 2% within 40 min. Altering the caveolin-binding-like motif of AT1-receptor thus affected the efficiency and the rate of internalization of 125I-Ang II.



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  		Figure 3. Functional properties of the AT1-YFFY/A receptor. A, Cells expressing the AT1 receptor ({circ}) or the AT1-YFFY/A receptor ({bullet}) were incubated with 125I-Ang II (0.1 nM) at 37 C for different periods of time. Internalization was stopped by washing the cells with PBS at 0 C. Acid-resistant binding was evaluated as indicated in Materials and Methods. Data are expressed as percent of total binding for each time point and represent mean ± SD of triplicate values. Internalization was calculated from the ratio of acid-resistant binding to total binding (acid-resistant plus acid-released). Nonspecific binding was measured in the presence of 1 µM unlabeled Ang II. These results are representative of three independent experiments. B, COS-7 cells expressing the AT1 receptor (0.26 picomol/mg protein) or the AT1-YFFY/A receptor (0.44 picomol/mg protein) were prelabeled for 20 h with 8 µCi/ml myo-[3H]inositol. Cells were then stimulated with 100 nM Ang II for 45 min. The incubation was stopped with perchloric acid and the inositol phosphates were measured as described in Materials and Methods. Results represent inositol phosphates (IP2 + IP3) produced over the basal level (4328 cpm for AT1 receptor and 3336 cpm for AT1-YFFY/A receptor) and expressed as the means ± SD of triplicate values. These results are representative of three independent experiments. C, Cells expressing the AT1 receptor were loaded with fura 2 and their [Ca2+]i was monitored upon stimulation with 100 nM Ang II and 1.2 µM ATP (acting through an endogenous purinergic receptor). The trace represents the average Ca2+ transients in four cells that responded to Ang II. D, Ang II- and ATP-induced Ca2+ transients in cells expressing the AT1-YFFY/A receptor (average response of four cells).

 
The functional properties of the transiently expressed receptors were further evaluated by measurement of Ang II-induced inositol phosphates production. Because the level of phospholipase C activation is directly related to the level of plasma membrane expression of the receptors, we reduced the amount of AT1 receptor cDNA in the transfection procedure to obtain a level of expression similar to that of the mutant AT1-YFFY/A receptor. In each experiment, the receptors densities were assessed by binding assay. Maximal activation (100 nM Ang II for 45 min) of COS-7 cells expressing the AT1 receptor caused an important accumulation of inositol phosphates (40,000 cpm above basal level) (Fig. 3BGo). Under the same conditions, COS-7 cells expressing the AT1-YFFY/A receptor had a much weaker response, accumulating only 10,000 cpm of inositol phosphates in 45 min. Despite their low production of inositol phosphates, cells expressing the AT1-YFFY/A receptor showed an Ang II-induced Ca2+ transient (Fig. 3DGo) alike that observed in cells expressing AT1 receptor (Fig. 3CGo). These results demonstrate that the fraction of mutated receptors reaching the plasma membrane is capable of triggering Ca2+ signaling.



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  		Figure 6. Regulation of AT1 receptor expression by DRIP78. Cos-7 cells expressing the AT1-receptor or the AT1-YFFY/A receptor (0.5 µg cDNA) were cotransfected with DRIP78 cDNA (2 µg). 125I-[Sar1,Ile8]Ang II binding was then evaluated on broken cells as indicated in Materials and Methods. Each point represents the mean of triplicate values (representative of three independent experiments).

 
Triton-based preparation of caveolae
To assess whether the AT1 receptor is present in caveolae, we used an established fractionation procedure based on the specific buoyant density of caveolin-rich membrane domains and their resistance to solubilization by the nonionic detergent Triton X-100 at 4 C (28). The presence of the receptor in the different fractions was evaluated by taking advantage of the fluorescence properties of the fusion proteins AT1-receptor-GFP and AT1-YFFY/A-receptor-GFP. As shown in Fig. 4AGo (open circle), with cells expressing the AT1-receptor-GFP, a minor proportion (approximately 8%) of the total fluorescence was recovered in the light fractions (fractions 4 and 5) of the gradient where caveolin was detected by Western blotting (Fig. 4CGo). This minor proportion did not change upon stimulation of the receptor with the agonist Ang II (filled circle). With cells expressing AT1-YFFY/A-receptor-GFP (Fig. 4BGo, open square), the profile of fluorescence distribution was very similar to that observed with AT1-receptor-GFP (Fig. 4BGo, filled square). The absence of the caveolin-binding-like motif did not modify the proportion of total fluorescence found in the light fractions of the gradient. Similar profiles of AT1 and AT1-YFFY/A receptors distribution were observed in Western blot assays with anti-GFP and anti-FLAG epitope antibodies (data not shown). These results suggest that there is no concentration of the AT1 receptor in the caveolae despite the presence of a caveolin-binding-like motif on the receptor.



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  		Figure 4. Triton-based preparation of caveolae. A, COS-7 cells transiently expressing the AT1-receptor-GFP were stimulated ({circ}) or not ({bullet}) with 100 nM Ang II for 5 min at 37 C. They were then homogenized in a medium containing 1% Triton X-100 as described in Materials and Methods. After sucrose gradient centrifugation, fractions were collected and their fluorescence level was evaluated. B, The same procedure was repeated with unstimulated COS-7 cells transiently expressing the AT1-receptor-GFP ({blacksquare}) or the AT1-YFFY/A-receptor-GFP ({square}). C, Sucrose gradient fractions were collected and subjected to immunoblot analysis with a specific antibody directed against caveolin-1. These results are representative of at least three independent experiments.

 
Detergent-free preparation of caveolae
It has been reported that the use of Triton X-100 in the initial homogenization step for the preparation of caveolae could cause the loss of certain caveolin-associated proteins (32). An alternative detergent-free approach was thus used to isolate caveolae. The same results were obtained when caveolae were prepared with the detergent-free approach. As shown in Fig. 5AGo, with cells expressing AT1-receptor-GFP (open circle), a small proportion of the total fluorescence was recovered in the light fractions of the gradient where caveolin-1 is localized (Fig. 5CGo). This proportion was not modified upon stimulation with Ang II (filled circle). With cells expressing AT1-YFFY/A-receptor-GFP, a very similar profile of fluorescence distribution was observed (Fig. 5BGo) with no noticeable change between the basal (open square) and the stimulated (filled circle) conditions.



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  		Figure 5. Detergent-free preparation of caveolae. A, COS-7 cells transiently expressing the AT1-receptor-GFP were stimulated ({circ}) or not ({bullet}) with 1 µM Ang II for 5 min at 37 C. They were then homogenized in a medium containing 500 mM sodium carbonate as described in Materials and Methods. After sucrose gradient centrifugation, fractions were collected and their fluorescence level was evaluated. B, The same procedure was repeated with COS-7 cells transiently expressing the AT1-YFFY/A-receptor-GFP stimulated ({square}) or not ({blacksquare}) with 1 µM Ang II for 5 min at 37 C. Basal fluorescence of cellular proteins was also evaluated with COS-7 cells transfected with pcDNA3 ({Delta}). C, Sucrose gradient fractions were collected and subjected to immunoblot analysis with a specific antibody directed against caveolin-1. These results are representative of at least three independent experiments.

 
Regulation of plasma membrane expression of the AT1 receptor by DRIP78
DRIP78 is an endoplasmic reticulum-localized protein regulating the intracellular routing of the D1 dopamine receptor through a specific interaction with a polyaromatic motif on the receptor (33). Because the polyaromatic motif of the D1 receptor has some similarity with the caveolin-binding-like motif of the AT1 receptor, we hypothesized that DRIP78 could influence the routing of the AT1 receptor. Figure 6Go shows that cell surface expression of the AT1 receptor increased significantly from 1.1 ± 0.2 pmol/mg of protein to 1.7 ± 0.2 pmol/mg of protein in cells coexpressing DRIP78. However, the cell surface expression of the mutant AT1-YFFY/A receptor was not significantly affected by the coexpression of DRIP78. Theses results suggest that the caveolin-binding-like motif is promoting a functional interaction between DRIP78 and the AT1 receptor.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Caveolae are plasma membrane-attached vesicular organelles that participate in many signal transduction-related events (24). Most proteins reported to be associated with caveolae contain a cytoplasmically accessible caveolin-binding motif, which is also present in the AT1 receptor. We therefore disrupted this motif in the C-terminal tail of the AT1 receptor and evaluated the functional properties of the mutant receptor. In binding experiments, the mutant receptor exhibited a high affinity for [Sar1, Ile8] Ang II but was relatively poorly expressed at the surface of COS-7 cells. Confocal microscopy further supported this observation by demonstrating an accumulation of the mutant-GFP fusion protein in an intracellular compartment. Our study shows the importance of the caveolin-binding-like motif in the cell surface expression of the AT1 receptor. It was previously reported by Gaborik et al. (34) that substitution of Phe309 for Ala caused a 5-fold decrease in AT1 receptor expression. Related studies in which another residue of the caveolin-binding-like motif, Tyr302, was substituted for Ala did not report any important change in cell surface expression of the AT1 receptor (26, 31). With a similar approach, although in a different cell type, Thomas et al. (35) observed that the substitution of Tyr302 for Ala caused a significant decrease in cell surface expression of the mutant AT1 receptor. They also observed a lower level of receptor expression after substituting Tyr312 for Ala. All together, these results demonstrate that several individual aromatic residues in the caveolin-binding-like motif of AT1 receptor can influence (at different degrees) the proper routing of the receptor to the plasma membrane. However, our results show that the most important effect was observed after disrupting the whole motif. These results show therefore that the caveolin-binding-like motif is important for proper routing of AT1 receptor to the cell surface. To our knowledge, these are the first results showing that the caveolin-binding-like motif plays a role in the routing of proteins to the plasma membrane.

Although the AT1-YFFY/A receptor was expressed at a low level at the cell surface, this level was nevertheless high enough to allow the assessment of the functional properties of the receptor. Agonist-induced internalization rate of the AT1-YFFY/A receptor was slower than that of the AT1 receptor. Moreover, the maximal level of internalization of the AT1-YFFY/A receptor was also affected. Clearly, the caveolin-binding-like motif plays an important role in the internalization of the AT1 receptor. Hunyady et al. (36) previously demonstrated that the sequence Ser335-Thr336-Leu337 in the cytoplasmic tail of AT1 receptor was also critical for agonist-induced internalization. With deletion mutants, Thomas et al. (35) identified two distinct regions in the cytoplasmic tail (region 315–329 and region 334–359 comprising the STL sequence mentioned above) involved in agonist-induced internalization of AT1 receptor. In that same study, Thomas et al. (35) reported that two mutants Y302A and Y312A had slightly reduced rates of internalization compared with the wild-type receptor. We also previously reported that substitution of Tyr302 for Ala slightly reduced the internalization rate of the AT1 receptor (26). Tyr302 and Tyr312 belong to the caveolin-binding-like motif but their role in the internalization process was relatively minor compared with the striking effect reported here of disrupting the whole caveolin-binding-like motif. Interestingly, it appears that several different domains within the cytoplasmic tail of AT1 receptor are involved in its internalization. Our results provide the first evidence that the caveolin-binding-like motif plays an important role in the agonist-induced internalization of a GPCR.

We showed that the AT1-YFFY/A receptor was less efficient than the AT1 receptor to activate phospholipase C. We and others (30, 31, 37) previously demonstrated that the extent of phospholipase C activation is directly related to the level of receptor expression. Therefore, in the study presented here, the transfection conditions were adjusted so that the level of expression of the mutant and the wild-type receptors were identical. Under these conditions, the AT1-YFFY/A receptor was 4-fold less efficient than the AT1 receptor at activating phospholipase C. These results clearly demonstrate that the caveolin-binding-like motif in the cytoplasmic tail of the AT1 receptor is required for efficient activation of phopholipase C. It was previously reported that some residues belonging to the caveolin-binding-like motif of the AT1 receptor are involved in the activation of phospholipase C. Ohyama et al. (12) showed that a mutant AT1 receptor in which amino acid residues 309–359 were deleted was a weak activator of phospholipase C. A report by Sano et al. (13), showed that a single point mutation (Y312A) considerably reduced the capacity of the AT1 receptor to activate phospholipase C. Other studies showed that mutation of Tyr302 significantly reduced AT1 receptor-induced phospholipase C activation (26, 31, 38). A synthetic peptide corresponding to the sequence 304–316 of the AT1 receptor was shown to reduce by 70% Ang II-induced Gq activation (39). All these results are compatible with a role of the caveolin-binding-like motif in phospholipase C activation.

The caveolin-binding motif is known as a polyaromatic docking site recognizing the polyaromatic scaffolding domain of caveolin, thus stabilizing the association of proteins to caveolae (16). The presence of such a motif within the AT1 receptor and its important functional role were suggesting that the receptor could concentrate in caveolae. Unexpectedly, under our experimental conditions, the proportion of AT1 receptor found in caveolae was not very high and this proportion was not modified after stimulation with Ang II. Not surprisingly, the AT1-YFFY/A receptor (which does not contain the caveolin-binding-like motif) was not either found in high proportion in caveolae. In contrast to our results, Ishizaka et al. (40), previously observed some translocation of the AT1 receptor to caveolae after stimulation of smooth muscle cells with Ang II. This discrepancy could be related to cell type specificities. Interestingly, of the three known isoforms of caveolin, caveolin-3 is exclusively expressed in muscle cells, and its presence was detected in smooth muscle cells of the arterial vasculature (41). Although it remains to be shown that AT1 receptor selectively interacts with caveolin-3, it is tempting to propose that such an interaction could be responsible for the translocation of AT1 receptor to caveolae during smooth muscle cell activation with Ang II. In COS-7 cells, we did not observe any agonist-induced translocation of AT1 receptor to caveolae. What then could be the role of the caveolin-binding motif ? We would like to suggest that this motif may serve as a docking site for interaction with undefined accessory protein(s). In support of this suggestion, it was recently shown that a novel membrane-associated endoplasmic reticulum protein (DRIP78) interacts with a polyaromatic motif in the C-terminal tail of the dopamine D1 receptor and regulates its transport to the plasma membrane (33). In the study presented here, we demonstrated that overexpression of DRIP78 increased the cell surface expression of AT1 receptor, an effect that was not observed with the AT1-YFFY/A receptor. These results suggest that the AT1 receptor may interact with DRIP78 through its caveolin-binding-like motif. Further studies are needed to show direct interaction between both proteins and to identify other putative accessory proteins that could interact with the AT1 receptor or other GPCRs through a caveolin-binding-like motif and regulate their functional properties.

In conclusion, we have shown that the caveolin-binding-like motif on the AT1 receptor is involved in its routing to the plasma membrane, in its efficiency to activate phopholipase C and in its endocytosis. These effects were produced under conditions where the AT1 receptor was not concentrated in caveolae. Our results rather suggest that the caveolin-binding-like motif may promote the interaction of the AT1 receptor with accessory proteins involved in the regulation of its expression and functions.


   Acknowledgments
 
We thank Dr. Leonid Volkov for his technical assistance in confocal microscopy studies and helpful discussions. We also thank Dr. Qun-Yong Zhou for his generous gift of DRIP78 cDNA.


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

Abbreviations: Ang II, Angiotensin II; AT1 receptor, type 1 receptor for Ang II; Bmax, maximum binding capacity; DRIP78, dopamine receptor interacting protein of 78 kDa; FBS, fetal bovine serum; GFP, green fluorescent protein; GPCR, G protein-coupled receptors; HBSS, Hanks’ balanced salt solution; Kd, dissociation constant; MBS, 2-[N-morpholino]ethanesulfonic acid-buffered saline.

Received July 5, 2002.

Accepted for publication September 4, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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