Intracellular Trafficking of Angiotensin II and its AT1 and AT2 Receptors: Evidence for Selective Sorting of Receptor and Ligand

Lutz Hein, Lorenz Meinel, Richard E. Pratt, Victor J. Dzau and Brian K. Kobilka

Falk Cardiovascular Research Center and Department of Medicine (L.H., R.E.P., V.J.D., B.K.K.) Howard Hughes Medical Institute (B.K.K.) Stanford University School of Medicine Stanford, California 94305
Department of Pharmacology (L.H., L.M.) University of Wuerzburg Wuerzburg, Germany
Department of Medicine (R.E.P, V.J.D.) Brigham and Women’s Hospital Harvard Medical School Boston, Massachusetts 02115


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Angiotensin II (Ang II) binds to two different receptor subtypes, AT1 and AT2 receptors. In many cases, receptor stimulation by Ang II is followed by a rapid desensitization of the intracellular signal transduction and a decrease in cell surface receptor number. The present study was designed to examine by immunofluorescence microscopy the cellular trafficking pathways of Ang II and its AT1a and AT2 receptors in human embryonal kidney 293 cells stably expressing these receptor subtypes. Fluorescently labeled Ang II and AT1a receptors were rapidly internalized into endosomes. AT2 receptors were localized in the plasma membrane and did not undergo endocytosis upon agonist stimulation. After removal of agonist, AT1a receptors recycled to the plasma membrane, whereas fluorescently labeled Ang II was targeted to the lysosomal pathway. Even though no further loss of surface receptor was measurable by ligand binding at steady state, fluorescein-Ang II was continuously internalized, and cycling of receptor between endosomal vesicles and the plasma membrane was detected by antibody feeding. These experiments provide evidence for subtype-specific receptor sorting and internalization of Ang II and its AT1a receptor as a receptor-ligand complex, and they suggest that the sequestration of receptors into endosomes is in dynamic equilibrium with receptor cycling to the plasma membrane. Finally, internalization of AT1a receptors and Ang II persists after desensitization mechanisms have attenuated Ca2+ and inositol 1,4,5-trisphosphate signaling.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Angiotensin II (Ang II) is the effector peptide of the renin-angiotensin system, which plays a major role in the regulation of cardiovascular homeostasis. The biological actions of Ang II are quite diverse, mediating contractile activity in vascular smooth muscle, aldosterone release in the adrenal gland, and growth-modulating effects on smooth muscle cells and cardiac myocytes (1). In its target cells, Ang II binds to different subtypes of G protein-coupled receptors. Most of the cardiovascular actions of Ang II have been attributed to stimulation of the AT1 receptor subtype, which has been cloned from different species (2, 3, 4). A second receptor subtype, the AT2 receptor, which is highly expressed in fetal tissues, has been cloned recently (5, 6). Inactivation of the AT2 receptor by gene targeting in mice suggests that this receptor subtype may also participate in blood pressure regulation and in central nervous functions of Ang II (7, 8).

The cardiovascular effects of Ang II are frequently subject to rapid desensitization. For other types of G protein-coupled receptors, different mechanisms of desensitization have been characterized (9). These processes include 1) phosphorylation of specific intracellular receptor sites resulting in uncoupling from the G protein, 2) sequestration of receptors into endosomal vesicles, and 3) down-regulation of the total receptor number of a cell. The contribution of receptor sequestration to the desensitization process is still unclear. Tachyphylaxis of vascular contractile responses to Ang II has routinely been attributed to loss of cell surface receptors by sequestration (10, 11). Previously, indirect approaches have been used to study the internalization of angiotensin receptors. Sequestration of AT1 receptors has been observed as a rapid loss of cell surface ligand-binding sites accessible to radiolabeled derivatives of Ang II (11, 12, 13, 14). In addition, various cell types have been shown to internalize [125I]Ang II (12) or an Ang II-colloidal gold conjugate (15, 16). From the observation that Ang II was taken up by endocytosis, and angiotensin receptors were sequestered intracellularly, it was concluded that Ang II and its receptor were internalized as a receptor-ligand complex (14). However, due to the technical limitations of this approach, it was not possible to distinguish between the intracellular trafficking routes of Ang II and its receptors.

In the present study, we combined immunofluorescence staining of angiotensin receptors and fluorescein labeling of Ang II to follow the intracellular trafficking of Ang II and its AT1a and AT2 receptor subtypes by immunofluorescence microscopy. These experiments provide evidence for the internalization of Ang II and its AT1a receptor as a receptor-ligand complex, and they suggest that the receptor-ligand complex dissociates after endocytosis, with the receptors recycling to the plasma membrane and Ang II being transported to lysosomes. Sequestration of AT1a receptors into endosomes is in dynamic equilibrium with receptor recycling to the plasma membrane and continues after desensitization mechanisms have effectively attenuated the Ca2+ and inositol 1,4,5-trisphosphate (IP3) signaling pathways. The redistribution of receptor after agonist exposure is subtype specific, as the AT2 receptor does not undergo endocytosis.


    RESULTS AND DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Properties of Epitope-Tagged Angiotensin Receptors and of Fluorescein-Labeled Ang II
For detection of the trafficking pathways of the angiotensin receptor subtypes, we used the mouse AT1a and AT2 receptors that were epitope tagged (flag epitope DYKDDDD) (17) at the extracellular amino-terminus (Fig. 1AGo) and fluorescein-labeled Ang II (Fig. 1BGo). The flag epitope has been previously used to study the intracellular trafficking pathway of the thrombin receptor (18, 19), gastrin-releasing peptide receptor (20), and TRH receptor (21). Wild type and flag epitope-tagged receptors were stably expressed in human embryonal kidney 293 cells, and their ligand binding and signal transduction properties were determined. The ligand binding properties of the epitope-tagged receptor were indistinguishable from those of the wild type AT1a receptor (Fig. 1AGo). Similarly, stimulation of the flag-tagged receptor with Ang II induced an increase in IP3 (Fig. 1CGo) and intracellular Ca2+ levels (Fig. 1DGo). Under these conditions the cells were refractory to further stimulation by Ang II (Fig. 1DGo), indicating desensitization of the flag-AT1a receptor. The time course and magnitude of Ang II-induced changes in intracellular Ca2+ and IP3 were identical for flag-tagged and wild type AT1a receptors (data not shown). Similar to the AT1a receptor, flag epitope tagging of the AT2 receptor subtype at the amino-terminus did not change expression levels or ligand binding characteristics of flag-AT2 compared with those of the wild type AT2 receptor after transfection into COS-7 or 293 cells (data not shown).



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Figure 1. Characterization of the Epitope-Tagged Angiotensin AT1a Receptor and Fluorescein-labeled Angiotensin II

A, Wild type and flag-tagged AT1a receptors were stably expressed in human 293 cells, and radioligand binding profiles were determined on membrane preparations. Displacement binding curves for Ang II, the AT1 antagonist PD134756, and the AT2 receptor antagonist PD123319 did not differ between wild type (open symbols) and flag-tagged (closed symbols) angiotensin AT1a receptors. B, Displacement of [125I]Sar-Ile-Ang II by FITC-Ang II at the AT1a receptor. Fluorescein labeling of Ang II did not change its binding properties to the AT1a receptor. C, Intracellular IP3 levels in 293 cells stably expressing flag-tagged AT1a receptors. Stimulation of cells with 100 nM Ang II results in a transient increase in intracellular IP3 concentration, which rapidly returns to baseline values. D, Ang II-induced increase in cytoplasmic calcium in 293 cells stably expressing flag-tagged AT1a receptors. Cells were loaded with fluo-3/AM and analyzed by confocal, time lapse fluorescence imaging as described in Materials and Methods. After the initial stimulation with 100 nM Ang II, the cells were refractory to a second Ang II (100 nM) stimulus. Iono, 15 µM ionomycin; EGTA, 1 mM EGTA.

 
To allow fluorescence detection of the receptor agonist, Ang II was labeled at its amino-terminus with fluorescein isothiocyanate (22, 23). Addition of the fluorescent label to the angiotensin peptide did not significantly change its affinity for the flag-tagged AT1a receptor as determined by radioligand membrane binding (Fig. 1BGo). In addition, fluorescein isothiocyanate-labeled angiotensin II (FITC-Ang II) retained its agonist properties, as it was able to stimulate the production of IP3 (data not shown) and induce agonist-dependent endocytosis of AT1a receptors (see below) to a similar extent as unlabeled Ang II.

Ang II Induces Internalization of AT1a Receptors but not of AT2 Receptors
Confocal laser scanning microscopy was used to visualize the cellular localization of AT1a and AT2 receptors after immunostaining. In unstimulated human 293 cells stably expressing flag-tagged AT1a receptors, the AT1a subtype was detected in the plasma membrane by M1 antibody staining of nonpermeabilized cells (Fig. 2AGo). No antibody binding was detectable in untransfected 293 cells (data not shown). In addition to its surface localization, a smaller amount of AT1a receptor was found in intracellular vesicles in permeabilized cells (Fig. 2BGo). Flag-tagged AT2 receptors were localized on the cell surface, and no intracellular AT2 receptors could be detected (Fig. 2CGo). The presence of an intracellular pool of receptor has been observed for other G protein-coupled receptors, including the {alpha}2c-adrenergic receptor (24) and the thrombin receptor (19). In the case of the thrombin receptor, there is evidence that the intracellular pool of receptor serves as a reservoir of receptor protected from thrombin cleavage and activation (19). The functional role of intracellular AT1a and {alpha}2c-adrenergic receptor remains to be determined.



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Figure 2. Cellular Localization of Angiotensin AT1a and AT2 Receptors Transfected into Human 293 Cells

Under steady state conditions, flag epitope-tagged AT1a receptors were detected on the cell surface of nonpermeabilized transfected 293 cells (A) and in an intracellular localization in permeabilized cells (B, arrowheads). Flag-tagged AT2 receptors were only localized in the plasma membrane of permeabilized cells (C). No positive staining was observed in untransfected cells incubated with the M1 anti-flag antibody. The open boxes on top of the figure indicate that images visualize AT1a receptors (A and B) or AT2 receptors (C). Bars = 10 µm.

 
To follow the internalization of cell surface AT1a receptors after agonist stimulation, cells expressing flag-tagged AT1a receptors were incubated with the M1 antiflag antibody at 37 C in the absence or presence of agonist before fixation and fluorescence staining (Fig. 3Go). Using this approach, it was possible to label the plasma membrane AT1a receptors with antibody (Fig. 3AGo) without labeling the intracellular pool of AT1a receptors, and the fate of plasma membrane receptors could be determined. AT1a receptors were internalized into small intracellular vesicles upon stimulation with 1 µM Ang II (Fig. 3BGo) or 1 µM FITC-Ang II for 10 min (Fig. 3CGo). Addition of the AT1-specific antagonist PD 134756 alone did not change the cellular distribution of receptors (Fig. 3DGo). Internalization of receptors induced by Ang II or FITC-Ang II was completely blocked in the presence of the antagonist PD 134756 (Fig. 3Go, E and F). The AT2 receptor subtype was localized on the cell surface in unstimulated cells (Fig. 3GGo), but it did not undergo endocytosis upon stimulation with Ang II (Fig. 3HGo) or FITC-Ang II (Fig. 3IGo). Even prolonged exposure of AT2 receptor-expressing cells to Ang II for up to 2 h did not result in a detectable redistribution of the cell surface receptors (not shown). This observation is consistent with radioligand binding experiments showing no sequestration of AT2 receptors after exposure of cells to Ang II (25, 26).



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Figure 3. Ang II Induces Internalization of AT1a Receptors, but not of AT2 Receptors

Flag-tagged AT1a receptors stably expressed in human 293 cells were labeled for 60 min with M1 antibody then incubated for an additional 10 min. Under control conditions (A), AT1a receptors were localized in the plasma membrane. 1 µM Ang II (B) or FITC-Ang II (C) caused internalization of receptor into intracellular vesicles (arrowhead). Addition of the AT1 antagonist PD 134756 alone did not change the surface localization of AT1a receptors (D), but the antagonist blocked agonist-induced internalization (E and F). In flag-tagged AT2 receptor-transfected 293 cells, AT2 receptors were detected on the cell surface in unstimulated cells (G) as well as in cells treated with Ang II (1 µM, 10 min; H) or FITC-Ang II (1 µM, 10 min; I). The open boxes on top of the figure indicate that images visualize AT1a receptors (A–F) or AT2 receptors (G–I). Bar = 10 µm.

 
Intracellular Pathways of Ang II and AT1a Receptors after Endocytosis
To follow the intracellular trafficking pathways of Ang II and the AT1a receptor, cells were incubated in the presence of fluorescent endocytosis markers. Texas Red-labeled transferrin (TR-transferrin) was used to visualize endosomal trafficking, Texas Red-labeled ovalbumin (TR-ovalbumin) was used to trace the lysosomal pathway. Transferrin and its receptor are constitutively internalized into endosomes and rapidly recycle to the cell surface (27). After 10 min of agonist stimulation, internalized AT1a receptors were partially colocalized with endocytosed, TR-transferrin, suggesting that AT1a receptors are internalized into endosomes (Fig. 4Go, A and B). The presence of internalized AT1a receptors in endosomes was further confirmed by colocalization with the transferrin receptor using a monoclonal antibody against the human transferrin receptor (data not shown). Internalization of the AT1a receptor was reversible upon removal of the agonist. If cells were stimulated with Ang II for 10 min and then incubated in fresh medium for 50 min to remove Ang II, AT1a receptors recycled to the plasma membrane (Fig. 4CGo, arrow), whereas TR-transferrin remained in intracellular vesicles after removal of Ang II (Fig. 4DGo).



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Figure 4. Endocytosis and Sorting of AT1a Receptors and FITC-Ang II

For detection of cell surface AT1a receptors, cells were prelabeled with M1 antibody for 30 min at 37 C, followed by incubation with 1 µM Ang II and 15 µM TR-transferrin. After 10 min of stimulation, receptor-positive vesicles (A, arrowhead) contained TR-transferrin (B, arrowhead) as well. The internalization of AT1a receptor was reversible, as incubation of cells in fresh medium containing TR-transferrin, but no Ang II, for 50 min, resulted in recycling of AT1a receptors to the plasma membrane (C, arrow), whereas transferrin receptors were still detected in intracellular vesicles (D). When cells were labeled with 1 µM FITC-Ang II and 15 µM TR-transferrin for 10 min, FITC-Ang II and transferrin were colocalized in intracellular vesicles (E and F). However, 50 min after removal of the FITC-Ang II from the medium, FITC-Ang II was still localized in vesicles (G). Only some of these vesicles were overlapping with the distribution of transferrin (G and H, arrowhead). The open boxes on top of the figure indicate that images visualize AT1a receptors (A and C) or FITC-Ang II (E and G) or TR-transferrin (B, D, F, and H). Bar = 7 µm.

 
To follow the fate of the ligand after receptor stimulation, 293 cells were exposed to fluorescently labeled Ang II. In cells stably transfected with the AT1a receptor, FITC-Ang II was rapidly internalized into transferrin-containing vesicles (Fig. 4Go, E and F). The endocytosis of FITC-Ang II was AT1a receptor mediated, as it could not be detected in the presence of the receptor antagonist PD 134756, in cells transfected with AT2 receptor, or in untransfected cells (data not shown). However, upon removal of the agonist for 50 min following the initial 10-min exposure to FITC-Ang II, the fluorescent angiotensin remained in intracellular vesicles (Fig. 4GGo). Only part of these FITC-Ang II-containing vesicles were transferrin positive (Fig. 4Go, G and H, arrowhead), suggesting that the intracellular trafficking pathways of FITC-Ang II and transferrin beyond the endosome are distinct.

Previous studies have demonstrated that an Ang II-colloidal gold conjugate can be internalized into lysosomal structures in vascular smooth muscle cells (15, 16). To test, whether the intracellular vesicles, which contain FITC-Ang II after prolonged labeling periods, are lysosomes, TR-ovalbumin was used as a marker for lysosomal targeting of endocytosed ligands. When 293 cells expressing AT1a receptors were incubated with TR-ovalbumin for 30–60 min, the fluorescent ovalbumin was detected in intracellular vesicles, which could be stained with an antiserum against the lysosomal membrane protein lgp-120 (28) (results not shown). After 60 min of exposure of cells to Ang II and TR-ovalbumin, AT1a receptors and TR-ovalbumin were partially separated in the cells, with AT1a receptor-positive vesicles appearing in the periphery of the cells (Fig. 5Go, A and C, arrowhead) and ovalbumin-containing vesicles in the periphery (Fig. 5BGo, arrowhead) and the center of the cells (Fig. 5Go, B and C, arrow). After removal of the agonist, AT1a receptors returned to the plasma membrane (Fig. 5Go, A and F, arrow), whereas the TR-ovalbumin remained in intracellular vesicles (Fig. 5Go, E and F, arrowhead).



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Figure 5. Recycling of AT1a Receptors and Lysosomal Targeting of FITC-Ang II after Endocytosis

Trafficking of AT1a receptors (upper panel) and FITC-Ang II (lower panel) was compared with the sorting of TR-ovalbumin (TR-Ov) as an endocytosis marker that is destined for lysosomal degradation. When AT1a receptors were stimulated with 1 µM Ang II for 60 min, they appeared in intracellular vesicles (A, arrowhead), which showed only a partial overlap with the endocytosed TR-ovalbumin (B and C, arrowhead). Under these conditions, the majority of endocytosed TR-ovalbumin is localized in vesicles that do not contain AT1a receptors (B and C, arrow). Part of these vesicles represent lysosomes, as delivery of TR-ovalbumin to lysosomes was confirmed by immunostaining with an antibody against the lysosomal membrane protein lgp-120 (not shown). Upon agonist removal for 50 min, AT1a receptors returned to the plasma membrane (D, arrow), whereas TR-ovalbumin retained its vesicular distribution (E and F, arrowhead). In contrast to the endocytosis and recycling of the AT1a receptor, FITC-Ang II showed significant overlap in its endocytosis pathway with TR-ovalbumin (G–I, arrowhead) and did not redistribute to the cell surface after agonist removal (K–M, arrowhead). The open boxes on top of the figure indicate that images visualize AT1a receptors (A and D) or FITC-Ang II (G and K) or TR-ovalbumin (B, E, H, and L). Bar = 7 µm.

 
When FITC-Ang II was used to monitor the intracellular fate of the agonist, the intracellular distribution of fluorescently labeled Ang II and ovalbumin overlapped (Fig. 5Go, G–I). After removal of FITC-Ang II from the media and washout for 50 min, both labels showed significant colocalization in intracellular vesicles (Fig. 5Go, K–M, arrowhead), indicating that FITC-Ang II was diverted to the lysosomal system. Taken together, these results suggest that AT1a receptors can recycle to the plasma membrane after endocytosis, and that Ang II itself is transported to lysosomes.

Continuous Endocytosis and Recycling of AT1a Receptors
Internalization of AT1 receptors can be detected by radioligand binding assay in a variety of cell types as a decrease in cell surface receptor number by 50–75% (10, 11, 29). This sequestration of AT1a receptor reaches an equilibrium after 20–30 min of agonist stimulation. The observation that AT1a receptors recycle to the plasma membrane after agonist removal suggests that this equilibrium might be a dynamic one, with continuous internalization and recycling of the receptor to the cell surface rather than a static equilibrium, where receptors do not recycle in the presence of agonist. To test this hypothesis, maximum internalization of AT1a receptors was induced by exposure of 293 cells to 1 µM Ang II for 30 min. Under this condition, no further decrease in cell surface receptor number could be observed (Fig. 6AGo). Receptor signaling through intracellular IP3 accumulation was already desensitized after 10 min of exposure to agonist (see Fig. 1CGo). Surprisingly, cells were able to internalize FITC-Ang II (Fig. 6BGo) as well as AT1a receptors labeled with M1 antiflag antibody (Fig. 6CGo) into intracellular vesicles, even after the maximum receptor internalization was reached. This phenomenon could be blocked by addition of the receptor antagonist PD 134756 (data not shown). This result demonstrates that endocytosis and recycling of receptors to the plasma membrane occur continuously even in the presence of receptor agonist. The internalization and recycling process of the AT1a receptor seems to be independent of receptor signaling, as receptor internalization and recycling could be detected even in the presence of agonist for up to 50 min. At this time, signal transduction by the AT1a receptor via IP3 (Fig. 1CGo) or Ca2+ pathways (Fig. 1DGo) was completely desensitized (29, 30). However, Ang II-stimulated diacylglycerol levels in vascular smooth muscle cells remain elevated even after 30- to 60-min exposure to Ang II, and it has been suggested that the sustained diacylglycerol accumulation is linked to the internalization of receptor-ligand complexes (10). In 293 cells stably expressing AT1a receptors, inhibition of protein kinase C by staurosporin or down-regulation of protein kinase C by prolonged treatment with the phorbol ester phorbol 12-myristate 13-acetate did not change the internalization of angiotensin receptors (29).



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Figure 6. Continuous Internalization and Recycling of Ang II and its AT1a Receptor

AT1a receptor-expressing 293 cells were stimulated for 30 min with 1 µM Ang II to induce maximal AT1a receptor endocytosis. Under these conditions, cell surface receptor number decreased to 40% of the initial value and reached a new equilibrium (A). In the continued presence of agonist, cells were incubated for 20 min with FITC-Ang II (B) or with anti-flag M1 antibody (C). In both cases, fluorescently labeled agonist (B; FITC-Ang II) and AT1a receptor (C) continued to be internalized into intracellular vesicles (B and C, arrowhead). The open boxes on top of the figure indicate that images visualize FITC-Ang II (B) or AT1a receptors (C). Bar = 5 µm.

 
Inhibition of Endosomal Acidification Inhibits Recycling of AT1a Receptors
As the cellular trafficking pathway of Ang II differs from the recycling pathway of its AT1a receptor, it is tempting to speculate that dissociation of ligand and receptor is necessary for recycling of the receptor to the plasma membrane. Binding of Ang II to its receptor is known to be very sensitive to acidic pH, as washing of cells in an acidic buffer is commonly used in internalization assays to distinguish between cell surface and intracellular receptors. To test whether interference with the endosomal acidification process would influence the recycling of the AT1a receptor, cells were treated with ammonium chloride or chloroquine, and recycling of receptor was monitored by immunofluorescence or enzyme-linked immunosorbent assay. For these experiments, receptor internalization was induced by 1 µM Ang II for 10 min (Fig. 7Go, A and B). Upon removal of Ang II and incubation of cells in normal medium, AT1a receptors recycled to the cell surface (Fig. 7AGo, open squares, and Fig. 7CGo). When quantitated by enzyme-linked immunosorbent assay, approximately 65% of the previously internalized AT1a receptors returned to the cell surface (Fig. 7AGo). Ammonium chloride and chloroquine significantly inhibited the recycling of AT1a receptors (Fig. 7Go, A, D, and E).



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Figure 7. Recycling of Internalized AT1a Receptors Is Blocked by Inhibitors of Endosomal Acidification

Cell surface AT1a receptors were labeled with M1 antibody for 30 min at 37 C and briefly washed in fresh medium, and receptor internalization was induced for 10 min with 1 µM Ang II (A and B). Surface AT1a receptor number was determined by enzyme-linked immunosorbent assay (see Materials and Methods). During the recovery period, receptors recycled to the plasma membrane (A and C). Addition of chloroquine (0.1 mM; D) or ammonium chloride (10 mM; E) during the washout period inhibited the receptor recycling (A, D, and E). The open box on top of the figure indicates that images visualize AT1a receptors (B–E). Bar = 5 µm.

 
The observation that internalized AT1a receptors and endocytosed fluorescein-labeled Ang II were found in transferrin-containing endosomes, suggests that AT1a receptors are endocytosed by the same pathway used by ß2-adrenergic receptors, which have been localized in coated pits and transferrin receptor-positive endosomes after agonist stimulation (31, 32). At present, it is not possible to distinguish whether dissociation of the receptor-ligand complex in endosomes is necessary before further sorting of ligand (to lysosomes) and receptor (recycling to the plasma membrane) can occur. It is tempting to speculate what might be the physiological role of the continuous internalization and recycling. In the case of the ß2-adrenergic receptors, recycling to the plasma membrane was found to be associated with receptor resensitization. It has also been hypothesized that angiotensin receptors may play a role in the clearance of Ang II from the plasma (33). In these studies, inhibition of the AT1a receptor by losartan in rats decreased the apparent MCR of Ang II. Interestingly, blockade of the AT2 receptor subtype had the opposite effect (33).

In summary, we have provided experimental evidence that 1) Ang II causes a subtype-specific endocytosis of AT1a receptors but not AT2 receptors; 2) internalization and recycling of the AT1a receptor are dynamic processes and continue even after IP3 and Ca2+ signaling pathways have been desensitized; and 3) dissociation of the internalized receptor-ligand complex in endosomes is necessary for recycling of the AT1a receptor to the plasma membrane and further trafficking of Ang II.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Receptor Constructs and Expression
For immunofluorescence detection, the mouse angiotensin AT1a receptor (4) and the mouse AT2 receptor were epitope tagged at the amino-terminus or the carboxyl-terminus of the receptor. The amino-terminal epitope contained a modified influenza hemagglutinin-cleavable signal sequence (MKTIIALSYIFCLVFA) followed by the flag epitope (sequence DYKDDDD) (19). The monoclonal M1 antibody (Eastman Kodak, New Haven, CT) recognized the DYKDDDD sequence in the engineered receptors only, if the signal sequence was properly processed and cleaved at the predicted signal peptidase site (17, 34). To control for potential adverse effects of the flag epitope on receptor function, the AT1a receptor was tagged at its intracellular carboxyl-terminus with the influenza hemagglutinin epitope 12CA5 (sequence YPYDVPDYA) (35). This epitope is recognized by the monoclonal 12CA5 antibody (Berkeley Antibody Co., Berkeley, CA). The epitopes were integrated into the complementary DNA (cDNA) encoding the mouse angiotensin AT1a or AT2 receptors by oligonucleotide-directed mutagenesis using the PCR. Mutations were confirmed by dideoxy sequencing (Sequenase, U.S. Biochemical Corp., Cleveland, OH) using the chain termination method. For expression in cells, epitope-tagged AT1a receptor cDNAs were subcloned into the eukaryotic expression vector pBC12MI and cotransfected with pSV2-neo into human embryonal kidney HEK 293 cells by calcium phosphate precipitation. Flag-tagged AT2 receptor sequence was subcloned into the pcDNA3 vector for expression in 293 cells. Stable transfectants were selected with G418 and screened for receptor expression by immunofluorescence staining. Cells were maintained in high glucose DMEM containing 10% FCS (HyClone, Logan, UT; Life Technologies), gentamicin (100 µg/ml), and 0.8 mg/ml G418 geneticin (Life Technologies). Untransfected 293 cells were kept in the same medium without addition of geneticin.

Fluorescent Labeling of Ang II
Ang II was labeled at the amino-terminus with FITC as described previously (22). Briefly, 1 µmol Ang II was incubated with 1 µmol FITC in 100 mM NaHCO3, pH 8.5, including 25% acetone. After 2 h, the coupling reaction was stopped by adding 10 µmol glycine in 100 mM NaHCO3, pH 8.5. FITC-Ang II was separated from unbound FITC by chromatography on a Sephadex G-10 column. Experimental results obtained using Ang II that was FITC labeled with this method were identical to those obtained with commercially available FITC-Ang II (RBI Research Biochemicals International, Natick, MA).

Radioligand Binding
Receptor binding assays were performed as described previously (4). Briefly, transiently transfected COS-7 cells or stable 293 cell transfectants were lysed in 10 mM Tris-HCl (pH 7.4) and 1 mM EDTA buffer and scraped off plates. Membrane homogenates were prepared as previously described (4). Binding assays were performed in 500 µl buffer (75 mM Tris-HCl, 1 mM EDTA, 12.5 mM MgCl2, and 0.1% BSA, pH 7.4) for 1 h at 22 C. Saturation isotherms were obtained by incubating membranes with varying concentrations of [125I]Sar-Ile-Ang II (2200 Ci/mmol; DuPont-New England Nuclear, Boston, MA; Amersham, Arlington Heights, IL). Nonspecific binding was determined by the addition of 10 µM unlabeled Ang II. Competition experiments were performed in the presence of varying concentrations of Ang II (Sigma Chemical Co., St. Louis, MO) or the AT1 and AT2 receptor-specific antagonists PD 134756 (identical with DuP 753, losartan) and PD 123319 (kindly provided by Parke Davis, Detroit, MI). Radioactivity bound to membranes was separated from free ligand by filtration through GF/C filters (Whatman, Clifton, NJ). Binding data were analyzed by nonlinear regression using InPlot software (GraphPad Software, San Diego, CA).

Measurement of Intracellular IP3 Levels
293 cells expressing AT1a receptors were grown on six-well culture dishes (Falcon) and stimulated with 100 nM Ang II in DMEM for 30 sec to 50 min. Reactions were stopped by adding 1 ml ice-cold 20% trichloroacetic acid (wt/vol) and extracted with water-saturated diethyl ether. The concentration of IP3 in these samples was determined using a competitive radioligand receptor binding assay (Amersham) (36). Results shown are the mean ± SEM from triplicate determinations of four independent experiments.

Intracellular Calcium
To measure changes in intracellular calcium levels under conditions identical to those used for immunofluorescence staining, cells on coverslips were loaded with the long wavelength calcium indicator fluo-3 as described previously (19). Briefly, fluo-3 acetoxymethylester (fluo-3/AM) was dissolved in 20% pluronic F-127 in dimethylsulfoxide and was diluted 1:100 in DMEM before use. Cells on coverslips were loaded with 5 µM fluo-3/AM for 30 min at 37 C, rinsed twice in fresh medium, and incubated for 15 min to allow deesterification. Coverslips with attached cells were mounted in a perfusion chamber in PBS for inspection in a Sarastro Phoibos 1000 confocal laser scanning microscope (Molecular Dynamics, Sunnyvale, CA). Images were recorded at 8-bit resolution using the fluorescein channel in time lapse mode in 5-sec intervals (objective, x20; 256 x 256 pixels). Cells were stimulated with 100 nM Ang II or 15 µM ionomycin. Fluorescence intensity was measured by placing circular spots over the widest part of any given cell, and average intensity was determined for a minimum of 20 cells/coverslip. For each experiment, six coverslips were measured under identical conditions, and experiments were repeated at least three times. The data displayed indicate the mean ± SE.

Immunofluorescence Microscopy
Two days before the experiments, cells were split on glass coverslips. After various treatments, cells were fixed in 4% paraformaldehyde as described previously (18, 19). For experiments using permeabilized cells, fixed specimens were incubated for 30 min in blocking buffer containing 0.2% Nonidet P-40, 5% nonfat dry milk, and 50 mM HEPES (pH 7.6). Subsequently, primary antibodies (final concentrations: M1 antiflag antibody, 10 µg/ml; 12CA5 antibody, 5 µg/ml) were applied in blocking buffer for 1 h. Secondary antibodies [goat antirabbit F(ab')2 fragment of IgG conjugated to Texas Red; Jackson ImmunoResearch, West Grove, PA; goat antimouse IgG FITC conjugate, Amersham) were diluted 1:500 and applied in blocking buffer. For selective detection of receptor antigen localized on the cell surface, nonpermeabilized cells were labeled with primary antisera in DMEM containing 10% FBS and 30 mM HEPES, pH 7.6, at 37 C. For labeling of the endocytic pathways, cells were incubated with TR-transferrin and TR-ovalbumin (Molecular Probes, Eugene, OR). Specimens were inspected by confocal laser scanning microscopy using a Sarastro Phoibos 1000 instrument or a Leica DM microscope equipped with Leica TCS confocal scanner (Leica, Deerfield, IL). Optical sections were scanned through cells 2 µm above the surface of the coverslip. Images were stored on optical laser disk and processed using Image Space (Molecular Dynamics, Mountain View, CA) and Adobe Photoshop 2.5.1 software (Adobe Systems, Mountainview, CA). For each experiment, three to five coverslips were treated and inspected under identical conditions, and experiments were repeated at least three times with identical results.

Enzyme-Linked Immunosorbent Assay of Cell Surface Receptor Antigen
To quantitate the amount of cell surface angiotensin AT1 receptors, stably transfected 293 cells were split onto 24-well plates (Falcon, Becton-Dickinson Labware, Franklin Lakes, NJ) at 5 x 104 cells/well. The next day, cells were washed with DMEM with 20 mM HEPES (pH 7.4) and 1 mg/ml BSA. Cells were incubated with M1 anti-flag antibody for 30 min at 37 C to label the epitope-tagged AT1 receptors on the cell surface. After a brief rinsing step to remove unbound M1 antibody, cells were incubated with 1 µM Ang II for 10 min to induce receptor internalization. To monitor recycling of internalized receptors previously decorated with the M1 antibody, cells were kept in fresh medium without Ang II for 50 min at 37 C. At the end of the experiment, cells were fixed for 5 min in freshly prepared 4% paraformaldehyde in PBS. Plates were washed with PBS and incubated with alkaline phosphatase-conjugated secondary antibody (Bio-Rad, Richmond, CA; 1:300 dilution in PBS-1% BSA). Plates were developed with alkaline phosphatase chromogenic substrate p-nitrophenylphosphate. OD405 was read after 20 min. Antibody binding data are expressed as specific binding (total minus nonspecific, with nonspecific being defined as the level of binding seen in untransfected 293 cells). Data shown are the mean ± SEM (n = 3) for a representative experiment of three performed.


    ACKNOWLEDGMENTS
 
The authors thank Drs. H. Sasamura and M. Mukoyama for providing the AT1a and AT2 receptor cDNAs.


    FOOTNOTES
 
Address requests for reprints to: Brian K. Kobilka, M.D, Howard Hughes Medical Institute, B 157 Beckman Center, Stanford University, Stanford, California 94305.

This work was supported in part by the Howard Hughes Medical Institute (to B.K.K.), NIH grants (to R.E.P. and V.J.D.), and a fellowship (to L.H.) from the German Research Foundation (Deutsche Forschungsgemeinschaft, Bonn).

Received for publication April 30, 1996. Revision received April 2, 1997. Accepted for publication May 9, 1997.


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