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 Womens Hospital Harvard
Medical School Boston, Massachusetts 02115
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ABSTRACT
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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.
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INTRODUCTION
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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.
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RESULTS AND DISCUSSION
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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. 1A
) and fluorescein-labeled Ang II (Fig. 1B
). 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. 1A
). Similarly, stimulation
of the flag-tagged receptor with Ang II induced an increase in
IP3 (Fig. 1C
) and intracellular Ca2+ levels
(Fig. 1D
). Under these conditions the cells were refractory to further
stimulation by Ang II (Fig. 1D
), 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.
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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. 1B
). 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. 2A
). 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. 2B
). Flag-tagged AT2 receptors
were localized on the cell surface, and no intracellular
AT2 receptors could be detected (Fig. 2C
). The presence of
an intracellular pool of receptor has been observed for other G
protein-coupled receptors, including the
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
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.
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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. 3
). Using
this approach, it was possible to label the plasma membrane
AT1a receptors with antibody (Fig. 3A
) 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. 3B
) or 1
µM FITC-Ang II for 10 min (Fig. 3C
). Addition of the
AT1-specific antagonist PD 134756 alone did not change the
cellular distribution of receptors (Fig. 3D
). Internalization of
receptors induced by Ang II or FITC-Ang II was completely blocked in
the presence of the antagonist PD 134756 (Fig. 3
, E and F). The
AT2 receptor subtype was localized on the cell surface in
unstimulated cells (Fig. 3G
), but it did not undergo endocytosis upon
stimulation with Ang II (Fig. 3H
) or FITC-Ang II (Fig. 3I
). 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 (AF) or AT2
receptors (GI). Bar = 10 µm.
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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. 4
, 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. 4C
, arrow), whereas TR-transferrin remained in intracellular
vesicles after removal of Ang II (Fig. 4D
).

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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.
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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. 4
, 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. 4G
). Only part of
these FITC-Ang II-containing vesicles were transferrin positive (Fig. 4
, 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 3060
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. 5
, A and C,
arrowhead) and ovalbumin-containing vesicles in the
periphery (Fig. 5B
, arrowhead) and the center of the cells
(Fig. 5
, B and C, arrow). After removal of the agonist,
AT1a receptors returned to the plasma membrane (Fig. 5
, A
and F, arrow), whereas the TR-ovalbumin remained in
intracellular vesicles (Fig. 5
, 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
(GI, arrowhead) and did not redistribute to the cell
surface after agonist removal (KM, 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.
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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. 5
, GI). After removal of FITC-Ang II
from the media and washout for 50 min, both labels showed significant
colocalization in intracellular vesicles (Fig. 5
, KM,
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 5075% (10, 11, 29). This
sequestration of AT1a receptor reaches an equilibrium after
2030 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. 6A
). Receptor signaling
through intracellular IP3 accumulation was already
desensitized after 10 min of exposure to agonist (see Fig. 1C
).
Surprisingly, cells were able to internalize FITC-Ang II (Fig. 6B
) as
well as AT1a receptors labeled with M1 antiflag antibody
(Fig. 6C
) 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. 1C
) or Ca2+ pathways (Fig. 1D
) 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.
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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. 7
, A and B). Upon removal of Ang II and
incubation of cells in normal medium, AT1a receptors
recycled to the cell surface (Fig. 7A
, open squares, and
Fig. 7C
). When quantitated by enzyme-linked immunosorbent assay,
approximately 65% of the previously internalized AT1a
receptors returned to the cell surface (Fig. 7A
). Ammonium chloride and
chloroquine significantly inhibited the recycling of AT1a
receptors (Fig. 7
, 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 (BE). Bar =
5 µm.
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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.
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MATERIALS AND METHODS
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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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